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gov/, a user friendly site.

clinical, and surgical research. Original studies must conform to
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On August 7, the Brazilian Senate passed a bill (180/2008)
establishing a quota policy for students’ admission to
federal universities. Beginning next year, Brazilian federal
universities must offer 50% of their places to students who
graduated from public schools. In each state, these places
must be distributed according to the state’s ethnic distribution (black or Brazilian natives). Although racial quota
policies rest on an image of social equality (and apart from a
philosophical discussion on reverse racism), this explicit
legal obligation is a serious breach of the constitutionally
established university autonomy. This policy transforms
Brazilian universities into a modern version of the Greek
Polyphemus myth.
Polyphemus was a giant Cyclops who lived in a cave on
the remote island of Cyclopes, where, according to Homer’s
Odyssey, Ulysses and his crew were shipwrecked after the
Trojan War. When Ulysses’ crew came upon this cave, it
was filled with sheep, which were the property of
Polyphemus. The son of Poseidon and Thoosa, this oneeyed monster’s tremendous strength contrasted with his
single large eye.
Universities are not unlike Polyphemus. Since their
creation, universities have been respected as centers of the
production and transmission of knowledge, and the purity
of this knowledge is guaranteed by universities’ independence and autonomy. University autonomy may be compared with the large eye of Polyphemus: it provides a broad
and critical view that oversees its scientific production and
prevents the interference of public and private interests.
Last year, an editorial published in Nature (1) addressed
the issue of how scientists are solving problems in highschool science education by engaging in actions such as
visits to high schools. To this approach, we added
comments on our experience at the State University of
Campinas, which has a high-school program in which
students visit research labs (2). We demonstrated that this

single-month placement of high-school students in our
laboratory presented science attractively and encouraged a
fair portion of the visiting students to seek admission to
good universities to pursue scientific careers. Furthermore,
our program, ’Vocations in Science and the Arts’, demonstrated that university initiatives can improve basic education without government interference.
To preserve a university’s autonomy, all rules involving
student admissions must be created by the university itself.
Government interventions, such as the recently approved
bill 180/2008 by the Brazilian Senate, may dramatically
interfere with this constitutionally guaranteed privilege and
may seriously damage academic activities. Because bill 180/
2009 was sanctioned by President Roussef, we understand
that its constitutionality must be challenged before the
Supreme Court.
Returning to the Polyphemus myth, when the giant
realized his cave had been invaded, he blocked its entrance
with a huge stone. Ulysses cleverly engendered a plan: he
offered Polyphemus strong wine, took advantage of the
giant’s drunkenness and destroyed his single eye. With no
vision, Polyphemus became defenseless, allowing Ulysses
and his men to steal the sheep and escape the cave and the
island.
We suggest that this decision by the Brazilian Senate to
force federal universities to reserve 50% of their admissions
for specific groups represents the wine designed to injure
the critical and observant eye of ‘‘Polyphemus’’. Are
Brazilian federal universities destined to become a blind
old mythical creature in the eyes of future generations?

Email: ward@fcm.unicamp.br
Tel.: 55 19 3521-9081
Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

CLINICS covers all medical areas. This editorial highlights the field of pulmonary research. We have selected
papers published from 2010-11 concerning the
Continuously Variable Rating concept, which we have
recently put forward as an alternative and hopefully
superior method of evaluating published scientific papers
(1).

bronchopulmonary dysplasia. Higher rates of patent ductus
arteriosus and indomethacin enhance these differences.
Schachner et al. (10) find that at reoperative levels
.502 ng/ml, the N-terminal fragment of pro-brain type
natriuretic peptide predicts mid-term mortality after isolated
coronary artery bypass grafting and is associated with
significantly higher hospital mortality and perioperative
complications. By measuring adequate discriminative power
and calibration, Vieira et al. (11) developed and validated a
predictive score for clinical complications during the intrahospital transport of infants treated in neonatal units. The
authors claim that this predictive score can help identify
infants at risk of clinical complications during intra-hospital
transports.

DIAGNOSTIC
Anciaes et al. (2) induced experimental emphysema in
BALB/c mice and found that morphometric parameters were
more reliable for detecting its presence than the functional
parameters measured with respiratory mechanics. Bosch et
al. (3) report that a quick diagnosis unit currently being used
in a Spanish public university hospital represents a useful
and cost-saving model for diagnosing patients with potentially severe diseases. Boskabady et al. (4) report that
carpentry work in the city of Mashhad (northeast Iran) was
associated with a high frequency of respiratory symptoms,
particularly after occupational exposure to irritating chemicals. Costa et al. (5) report that the pediatric risk of mortality
score showed adequate discriminatory capacity and thus
constitutes a useful tool for assessing the prognosis of
pediatric patients who have been admitted to tertiary
pediatric intensive care units. Faria et al. (6) report on using
a forced oscillation technique to investigate the mechanical
properties of the respiratory system to detect early smokinginduced respiratory involvement when pathological changes
are still potentially reversible; their findings support the use
of this technique as a versatile clinical diagnostic tool for
preventing, diagnosing and treating chronic obstructive lung.
Guimaraes et al. (7) report that a CT-guided percutaneous
fine needle aspiration biopsy of lung lesions had a lower
complication rate in their study, but the lesions that lacked
pleural contact had more complications. Pimenta et al. (8)
propose the desaturation distance ratio, a new composite
index that uses continuous peripheral oxygen saturation
(SpO2) instead of a walked distance (a six minute walk test),
as a more reliable tool for performing a functional evaluation
of interstitial lung disease. Rocha et al. (9) report that the
differences between renal function and the tubular handling
of potassium and phosphorus are present during the first
week of life in preterm neonates who will develop

ONCOLOGY
Ardengh et al. (12) report that using transesophageal
ultrasound-guided fine needle aspiration to investigate
mediastinal tumoral lesions is an alternative to surgical
procedures in a vast majority of cases. Miziara et al. (13)
report that single-photon emission computed tomography/
computed tomography with Tc-99m-sestamibi showed very
low sensitivity and accuracy for the nodal staging of
patients with non-small cell lung cancer, despite its high
level of specificity. Parra et al. (14) report a direct link
between low amounts of type V collagen and decreased cell
apoptosis, which may favor cancer cell growth in the Balb/c
mouse lung after chemical carcinogenesis. This result may
suggest that the strategies aimed at preventing decreased
type V collagen synthesis or local responses to reduced
apoptosis may have a greater impact on lung cancer control.
Pereira et al. (15) report that that even low levels of fine
particulate matter (PM2.5) increase the risk of urethaneinduced lung tumors in Swiss mice. Sardenberg et al. (16)
claim that lung metastasectomy is a safe and potentially
curative procedure for patients with treated non-lung
primary tumors. A select group of patients can achieve
long-term survival after lung resection. Terra et al. (17)
report that when performed on an outpatient basis in
patients with recurrent malignant pleural effusions and
Karnofsky Performance Status scores ,70, talc pleurodesis
is a safe and efficacious procedure that has a low
complication rate and helps avoid hospital admissions.
Zhang et al. (18) report that tumor-associated macrophages
in lung adenocarcinoma have an M2-polarized subtype and
are associated with poor prognoses, perhaps resulting from
accelerated lymphangiogenesis and lymph node metastasis.

Email: mauricio.silva@hc.fm.usp.br
Tel.: 55 11 2661-6235
Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.

VENTILATION
Casaroli et al. (19) report that a pneumoperitoneum
procedure, both alone and in combination with controlled

No potential conflict of interest was reported.

1237

Pneumological in Clinics
Rocha e Silva M

CLINICS 2012;67(11):1237-1240

ventilation, did not modify the peritoneal lymphatic
bacterial clearance in a rat bacterial peritonitis model.
Ferreira et al. (20) note that an elevated lower inflection
point and the sigmoidal shape of the pressure-volume
curves suggest that respiratory system compliance
decreased near the end-expiratory lung volume in idiopathic pulmonary fibrosis patients undergoing general
anesthesia and mechanical ventilation. Lopez et al. (21)
report that the need for more than two hours of mechanical
ventilation predicted bronchopulmonary dysplasia in preterm infants with a gestational age of .26 weeks. This
development could be an early marker for developing
bronchopulmonary dysplasia. Nery et al. (22) suggest that
daily screening to identify the patients who are able to
breathe without support is recommended to reduce the
length of mechanical ventilation. These authors also propose
noninvasive positive-pressure ventilation as a technique to
shorten the time that patients remain on invasive ventilation. They report that this intervention reduced the length of
invasive ventilation and total ventilatory support and
identify this technique as an independent factor associated
with survival. Schifelbain et al. (23) report no differences
between the Doppler echocardiographic variables and
electrocardiographic and other cardiorespiratory variables
during weaning from mechanical ventilation using pressure
support ventilation or the T-tube procedure. These authors
also report that the cardiac structures were smaller, the
isovolumetric relaxation time was longer, and the oxygenation level was greater in successfully weaned patients.

ALLERGY
Boskabady et al. (29) report the preventive effect of a
hydroethanolic extract of Nigella sativa on tracheal responsiveness and white blood cell count in the lung lavage fluid
of sensitized guinea pigs. Gomieiro et al. (30) report that a
respiratory exercise program increased muscle strength and
had a positive effect on patient health and quality of life in
older asthmatic adults. Therefore, a respiratory training
program could be included in the therapeutic approach in
older asthmatic adults. Guimaraes et al. (31) report on the
pulmonary function and prevalence of atopy in school-aged
children who had low birth weights as infants; no
significant differences were found in the lung functions of
the bronchopulmonary dysplasia patients and patients
without bronchopulmonary dysplasia, and no evidence of
an association was found between atopy and bronchopulmonary dysplasia.

INFECTOLOGY
Arslan et al. (24) report that plasma D-dimer levels, which
are directly related to the intra- and extra-vascular coagulation that occurs in acute and chronic lung damage in
patients with community-acquired pneumonia, increased
even for patients who did not have an accompanying
disease that would normally cause such an increase.
Capelozzi et al. (25) report a detailed histopathological
analysis of the open lung biopsy specimens from five acute
respiratory distress syndrome (ARDS) patients with confirmed H1N1 in which viral-like particles were successfully
observed (via an ultrastructural examination) in the lung
tissue. The bronchioles and epithelium rather than the
endothelium are most likely the primary targets of infection.
Chung et al. (26) report that after completing tuberculosis
treatment, several risk factors predict pulmonary function
deterioration and significant respiratory symptoms, and
multiple risk factors require pulmonary function tests to
monitor functional impairment progression, particularly
within the first 18 months after completing treatment.
Soeiro et al. (27) report that in autopsies of 4,710 patients
with acute respiratory failure, bronchopneumonia and
cancer were the two most common diagnoses. The most
prevalent pulmonary histopathological pattern was diffuse
alveolar damage, which was associated with different
inflammatory conditions. Toufen et al. (28) report that
despite the marked severity of lung disease at admission,
patients with acute respiratory distress syndrome (caused
by swine-origin influenza A virus infection) presented with
a late but substantial recovery over six months of follow-up.

APNEA
Neves et al. (34) evaluate the effects of sildenafil on the
autonomic nervous systems of patients with severe obstructive sleep apnea and suggest that in addition to worsening
sleep apnea, sildenafil may have immediate cardiac effects.
Romano et al. (35) report that flow limitation measurement
during wakefulness is a highly sensitive and reliable
method for identifying obstructive sleep apnea when the
test is positive and that this approach may reliably exclude
moderate and severe obstructive sleep apnea when the test
is negative.

COPD
Reis et al. (36) report that patients with chronic
obstructive pulmonary disease present with impaired
sympathetic-vagal balance at rest and that the cardiac
autonomic control of heart rate is associated with inspiratory muscle weakness in chronic obstructive pulmonary
disease. Silva et al. (37) find that respiratory alterations in
severe chronic obstructive pulmonary disease may be
identified through increased respiratory system impedance,
which is more evident in the expiratory phase. The authors
claim that these results confirm the potential of withinbreath analysis of respiratory mechanics for assessing
respiratory modifications related to chronic obstructive
pulmonary disease.

OBJECTIVE: To evaluate intraocular pressure in very low birth weight preterm infants and correlate it with
postconceptional age.
METHODS: The intraocular pressure in a prospective cohort of very low birth weight premature infants (defined as a
birth weight #1,500 g and gestational age #32 weeks) admitted to Hospital de Clı´nicas de Porto Alegre, Brazil was
evaluated weekly. The evaluated outcome was the variation in the intraocular pressure following changes in the
postconceptional age (defined as the gestational age at birth plus the age in weeks at the time of examination) in
the weeks following preterm birth. Mixed-effects models were used for the statistical analysis to determine the
intraocular pressure variation according to postconceptional age, and means and 10th and 90th percentiles were
calculated for the intraocular pressure values.
RESULTS: Fifty preterm infants with a mean gestational age of 29.7¡1.6 weeks and a mean birth weight of
1,127.7¡222.7 g were evaluated. The mean intraocular pressure for the entire cohort considering both eyes was
14.9¡4.5 mmHg, and 13.5% of all recorded intraocular pressure values were greater than 20 mmHg. The analysis
revealed a mean reduction in the intraocular pressure of 0.29 mmHg for each increase in postconceptional age
(p = 0.047; 95% CI: 20.58 to 20.0035). The mean intraocular pressure (P10–P90) decreased from 16.3 mmHg (10.52–
22.16) at 26.3 weeks to 13.1 mmHg (7.28–18.92) at 37.6 weeks of postconceptional age.
CONCLUSIONS: The mean intraocular pressure in very low birth weight preterm infants was 14.9¡4.5 mmHg. This
value decreased 0.29 mmHg per week as the postconceptional age increased.
KEYWORDS: Prematurity; Very Low Birth Weight Preterm Infants; Intraocular Pressure; Tonometry.
Lindenmeyer RL, Farias L, Mendonc¸a T, Fortes Filho JB, Procianoy RS, Silveira RC. Intraocular pressure in very low birth weight preterm infants and its
association with postconceptional age. Clinics. 2012;67(11):1241-1245.
Received for publication on April 6, 2012; First review completed on June 15, 2012; Accepted for publication on July 10, 2012
E-mail: jbfortes@cursohbo.com.br
Tel.: 55 51 9969 8081

diameter and the axial length of the eye and the postconceptional age (PCA) and birth weight. In 1999, Ricci (5) evaluated the IOP of 20 preterm infants in a longitudinal study
with five visits and concluded that the mean IOP following
birth decreased progressively.
Currently, more clinical information is available regarding
the IOP variation among VLBW preterm infants. In 2008, Ng
et al. (6) reported that the IOP decreases as the PCA increases
in preterm infants.
Our study aimed to evaluate the behavior of the IOP in
VLBW preterm infants, as well as its association with PCAs
up to 37.6 weeks.

INTRODUCTION
The survival rate for very low birth weight (VLBW)
preterm infants has increased since the 1950s as a result of
numerous advances in general perinatal care. The first
studies detailing the intraocular pressure (IOP) in this
population were published in the 1950s by Dolcet (1) and
Brockhurst (2), who found mean IOPs of 35 mmHg and
24.5 mmHg, respectively; both values were considered much
higher than those expected for normal adults. Thirty years
later, Musarella & Morin (3) re-examined the IOP in VBW
preterm infants and obtained isolated IOP values that could
be correlated with corneal diameter. Similarly, Tucker et al.
(4) identified the correlations between the IOP, corneal

METHODS
Study design

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.

This longitudinal study included VLBW preterm infants
(VLBW was defined as a birth weight [BW] #1,500 g; preterm
was defined as a gestational age [GA] #32 weeks) admitted to
the Hospital de Clı´nicas de Porto Alegre (HCPA) between 2008
and 2010.

No potential conflict of interest was reported.

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Intraocular pressure in preterm infants
Lindenmeyer RL et al.

CLINICS 2012;67(11):1241-1245

quiet and still to avoid a Valsalva-like effect. All the IOP
measurements were recorded by the same individual
(R.L.L.).

Setting
The study was conducted in the neonatal intensive care
unit (NICU) of HCPA, Porto Alegre, Brazil. HCPA is a
tertiary university hospital in an urban area with a population of approximately 3 million.

Statistical analysis
To detect a difference of 2.3 mmHg (¡2.0) with 90% study
power and a significance level of p,0.05, 16 patients (32 eyes)
were required. Neonatal data were evaluated using descriptive statistics and are reported as absolute values and
percentages. Mixed-effects models were used to analyze the
association between IOP and PCA. Percentiles (P10 and P90)
were used to describe the range of normality. For the
comparison of IOP and PCA, the level of significance was
set at 5% for a two-tailed sample distribution.

Patients
The study included VLBW preterm infants who were
admitted to the NICU. Exclusion criteria were ocular or
systemic malformations, genetic anomalies, death before 14
days of life, and infants with Stage III or IV intraventricular
hemorrhage. The gestational age of each infant was determined based on obstetric history and early obstetric ultrasound and confirmed by clinical examination of the newborn
infant. The PCA was defined the GA of the infant plus the
number of weeks since birth. All the infants were examined
while hospitalized in the NICU. Routine screenings for
retinopathy of prematurity (ROP) did not occur on the same
day that the IOP measurements took place.

Ethical issues
This study and its informed consent form were approved
by the Ethics and Research Committee of HCPA (n˚ 07-667).

RESULTS
Outcome evaluated

Between November 2008 and June 2010, 50 VLBW preterm
infants who met the inclusion criteria were examined. The
mean GA at birth was 29.7¡1.6 weeks (range: 26 to 32 weeks)
and the mean BW was 1,127.2¡222.7 grams (range: 710 to
1,500 g) for the entire cohort. All of the included patients
were also evaluated for ROP, and none of the infants
developed any stage of ROP.
The four planned evaluations to measure the IOP were
performed in 41 patients. Two patients underwent three
evaluations, one patient underwent two evaluations and six
patients underwent only one IOP measurement. The evaluations were missed because of prolonged clinical instability or
death. The mean time between birth and the first IOP
examination was 8.1¡5.4 days. A total of 356 (89%) IOP
measurements, out a total of 400 that were initially planned,
were performed (Table 1).
The mean IOP in both eyes for all measurements was
14.9¡4.5 (range: 6 to 27.7) mmHg. The mean RE IOP was
15¡4.3 (range: 6 to 27), and the LE IOP was 14.9¡4.8
(range: 6.3 to 27.7) mmHg. The IOP values were greater than
20 mmHg in 13.5% of the measurements. The mean IOP
values at timepoints 1, 2, 3 and 4 are shown in Table 1.
Table 2 shows the mean IOP values at each PCA week.
An analysis using mixed-effects models revealed a trend
towards a reduced RE and LE IOP as a function of PCA. In
the REs, the reduction was 0.29 mmHg for each one week

IOP.

Studied variables
The gestational age and BW were evaluated for the entire
cohort.

Intraocular pressure
Ophthalmological evaluations included IOP measurements at four weekly evaluations. Because of possible
cardiovascular and respiratory complications, all the evaluations were performed when the patient was clinically stable.
Subsequent evaluations were performed, preferably at
weekly intervals, and the PCA was adjusted at each examination date. All IOP measurements were obtained with the
infant lying supine and, when necessary, in the incubator.
After the application of anesthetic eye drops (0.5% proxymetacaine hydrochloride) in both eyes, a neonatal Barraquer
eyelid speculum was placed on the left eye (LE). The left eye
IOP was measured, and the procedure was repeated for the
right eye (RE). Three sequential IOP measurements, with 5%
confidence for each eye, were made using a previously
calibrated tonometer (Tonopen XLTM, Mentor O & O Inc.
Santa Barbara, CA, USA). The IOP measurement recorded for
each evaluation was the mean value of these three measurements. The measurements were taken when the infant was
Table 1 - Descriptive analysis of the evaluations.
Evaluation
1

study evaluated the behavior of the IOP in VLBW preterm
infants and its association with PCAs of up to 37.6 weeks
because we expected that preterm infants would have the
same IOP as term infants after that timepoint. Our findings
reflect the IOP values for prematurity for IOP examinations
performed before 37.6 weeks of PCA. To the best of our
knowledge, this value has not previously been described.
A study by Ng et al. (6) found an IOP reduction of
0.11 mmHg (p,0.001) for each one week increase in the
PCA. In their study, six IOP measurements were taken in
infants with PCAs that ranged from 26.1 to 46.4 weeks.
While our study found a greater reduction in IOP
(0.29 mmHg; p = 0.047), our subjects had a narrower PCA
range (26.3 to 37.6 weeks). This difference suggests that the
IOP reduction reported in the Ng et al. study may have been
greater because the infants were evaluated after 40 weeks
PCA, a point at which the IOP might not undergo any
additional changes. By contrast, our study determined the
IOP in infants younger than 37.6 weeks PCA; therefore, our
measurements were limited to infants who were actually
preterm. Our results may better reflect the behavior of the
IOP in the weeks following premature birth.
A previous study by Ricci (5) found that a reduced IOP
was associated with increases in the PCA and suggested this
was caused by the maturation of the aqueous drainage
system. Improvements in the aqueous flow coincide with
the complete formation of the aqueous drainage system.
Despite the differences in methods and statistical analyses,
our results agree with those of Ricci (5) and Ng et al. (6).
However, Musarella and Morin (3) did not find a direct
correlation between the IOP and PCA; they found that the
IOP decreased according to the increase in infant weight
and that the IOP might be associated with physical
development and maturity.
Historically, most studies of IOP in preterm infants used
only one isolated IOP measurement. The longitudinal
studies that found a negative correlation between IOP and
PCA were conducted by Ricci in 1998 and by Ng et al. in
2008. Ricci evaluated the IOP in 40 eyes of 20 premature
infants with GAs ranging from 26 to 32 weeks (mean:
29.5¡1.5 weeks) and used repeated measures ANOVA for

increase in PCA (p = 0.072; 95% CI, 20.6 to +0.026). In the
LEs, the reduction was 0.15 mmHg for each one week
increase in PCA (p = 0.42; 95% CI, 20.5 to +0.21). When both
eyes were analyzed together, the mean reduction was
0.29 mmHg for each one week increase in PCA (p = 0.047;
95% CI, 20.58 to 20.0035). Figure 1 shows the separate IOP
measurement values and their association with PCA. Lines
indicate the reduction in each eye and in both eyes. Based
on the correlation formula that we determined (y = 23.97 –
0.29x), the mean IOP varied according to the PCA from
16.4 mmHg at 26.3 weeks to 13.1 mmHg at 37.6 weeks.
Moreover, the variation between the 10th and the 90th
percentiles ranged from 10.5 to 22.2 mmHg at P10 and from
7.3 to 18.9 mmHg at P90 (Figure 2).

DISCUSSION
This study determined the IOP of VLBW preterm infants
using longitudinal measurements over four weeks. We found
that IOP of VLBW preterm infants, analyzed longitudinally,
was 14.9¡4.5 mmHg over a mean of four observations taken
four consecutive weeks after birth. Using mixed-effects
models, we observed a significant reduction in the IOP
(approximately 0.29 mmHg for each week of PCA). Our

Figure 2 - IOP values and their association with PCA. The lines represent the model that summarizes the association between the IOP
and PCA (blue = right eye; red = left eye).

We used a Tonopen XPTM tonometer because we have
found that it has a good correlation with Goldman/Perkins
tonometers. Our study found that the mean IOP in VLBW
preterm infants is 14.9 mmHg and that the IOP decreases as
the infant develops (by 0.29 mmHg per PCA week). Our
study found a greater reduction, 0.29 mmHg (p = 0.047),
over a narrow range of PCAs (26.3 to 37.6 weeks). Overall,
our study provides information regarding the behavior of
the IOP in VLBW premature infants. Further studies on this
subject are warranted to improve our understanding of the
IOP in this particular group of patients.

the statistical analysis of the IOP, although the IOP
measurements were not controlled for differences between
the various PCAs (5). By contrast, Ng et al. used mixedeffects models to analyze IOP measurements in 104 preterm
infants with a median (interquartile range) GA of 29.8 weeks
(range: 28.7–30.9 weeks) and a median BW of 1,208 g (range:
1,049–1,370 g) to adjust the IOP values to the corresponding
PCAs (6). In our study, the mean GA was 29.7¡1.6 weeks
(range: 26–32 weeks), and the mean BW was 1,127.2¡
222.7 g (range: 710–1,500 g) for the entire cohort.
Few studies have conducted similar investigations, and an
adequate method for studying this topic has rarely been
adopted. The best method for this type of study seems to be
longitudinal IOP measurements taken while the infant is
hospitalized to gain weight or to treat the many comorbidities
that may affect them after preterm birth. However, because
infants are born at different GAs and often have unstable
clinical conditions in the first weeks of life, it is difficult to
follow a protocol that meets all the requirements for the use
of repeated measures ANOVA to analyze the data. Therefore,
we chose to use mixed-effects models for statistical analysis
because it is more flexible for handling data and because it
can be used to evaluate both eyes at the same time.
To measure the IOP in newborn infants, we used a protocol
based on experiences in previous studies. Anesthetic eye
drops were mandatory to make the examination less
uncomfortable for the infant. Furthermore, the use of general
anesthesia would not be ethically correct and might affect
evaluations by reducing the IOP. A Barraquer eyelid
speculum was used because of the small size of the eye and
adnexa. There are no previous studies related to the
Barraquer eyelid speculum as a source of bias in IOP
measurements in preterm infants. However, a single study
(7) suggests that the use of the Alfonso eyelid speculum may
falsely elevate IOP by 4 mmHg in children 6 to 252 months of
age (mean: 70 months). Therefore, the use of the Barraquer
speculum could be one limitation of our study.

ACKNOWLEDGMENTS
The authors certify that the protocol for the research project has been
approved by a suitably constituted ethics committee in the institution
within which the work was undertaken and that the research protocol
conforms to the provisions of the Declaration of Helsinki in 1995 (as
revised in Edinburgh, 2000). The authors declare no financial support or
relationships that may pose a conflict of interest.

AUTHOR CONTRIBUTIONS
Silveira RC planned, conducted and revised all steps of the study and
approved the final manuscript. Lindenmeyer RL was the main investigator
and planned, conducted and collected all of the IOP measurements and
approved the final manuscript. Farias L organized the protocol and data
and approved the final manuscript. Mendonc¸a T organized the protocol
and data and approved the final manuscript. Fortes Filho JB was also a
main investigator and planned, conducted and revised all steps of the study
and approved the final manuscript. Procianoy RS planned, conducted and
revised all steps of the study and approved the final manuscript.

OBJECTIVES: To determine the frequency of medical adverse events in elderly patients admitted to an acute care
geriatric unit, the predictive factors of occurrence, and the correlation between adverse events and hospital
mortality rates.
METHODS: This prospective study included 171 admissions of patients aged 60 years and older in the acute care
geriatric unit in a teaching hospital in Brazil between 2007 and 2008. The following variables were assessed at
admission: the patient age, gender, number of prescription drugs, geriatric syndromes (e.g., immobility, postural
instability, dementia, depression, delirium, and incontinence), comorbidities, functional status (evaluated with the
Katz Index of Independence in Activities of Daily Living), and severity of illness (evaluated with the Simplified Acute
Physiology Score II). The incidence of delirium, infection, mortality, and the prescription of potentially inappropriate
medications (based on the Beers criteria) were assessed during hospitalization. An observer who was uninvolved in
patient care reported the adverse events.
RESULTS: The mean age of the sample was 78.12 years. A total of 187 medical adverse events occurred in 94
admissions (55%). The predictors of medical adverse events were undetermined. Compared with the patients with
no adverse events, the patients with medical adverse events had a significantly longer hospital stay (21.41¡15.08
days versus 10.91¡7.21 days) and a higher mortality rate (39 deaths [41.5%] versus 17 deaths [22.1%]). Mortality
was significantly predicted by the Simplified Acute Physiology Score II score (odds ratio [OR] = 1.13, confidence
interval [CI] 95%, 1.07 to 1.20), the Katz score (OR = 1.47, CI 95%, 1.18 to 1.83), and medical adverse events
(OR = 3.59, CI 95%, 1.55 to 8.30).
CONCLUSION: Medical adverse events should be monitored in every elderly hospitalized patient because there is no
risk profile for susceptible patients, and the consequences of adverse events are serious, sometimes leading to
longer hospital stays or even death.
KEYWORDS: Adverse Events; Elderly; Hospitalization; Risk Factor.
Szlejf C, Farfel JM, Curiati JA, Couto Junior EB, Jacob-Filho W, Azevedo RS. Medical adverse events in elderly hospitalized patients: A prospective
study. Clinics. 2012;67(11):1247-1252.
Received for publication on April 20, 2012; First review completed on June 3, 2012; Accepted for publication on July 2, 2012
E-mail: claujeru@gmail.com
Tel.: 55 11 8541-9196

one or more AEs in the hospital and approximately 50% of
these AEs may be preventable (1-9).
Hospitalized patients aged 65 years and older are at a
higher risk of AEs than young adults (1,2,5-8,10-15). The
incidence of AEs ranges from 5% to 58% in the elderly group
(1,2,4,5,11,12,16-19). AEs increase the burden of already
seriously ill elderly hospitalized patients and lead to
functional impairment or death in 5% to 27% of cases
(2,6,9,12,18,20). Patients who are injured as a result of medical
error spend more time in the hospital (7,8,10,11,14-16,20) and
have higher hospital costs (21,22). Several studies have
attempted to identify the risk factors that are associated with
the occurrence of AEs in hospitalized seniors, including the
length of the hospital stay, number of comorbidities,
admission to a psychiatric unit, severity of illness, level of
consciousness, number of drugs prescribed, and functional
status at the time of admission (11,12,16-18,20).

INTRODUCTION
An adverse event (AE) is generally described as an
unintended injury that 1) is caused by medical management
rather than a disease process and 2) results in death, a life
threatening illness, a disability at the time of discharge, an
admission to the hospital, or prolongation of the hospital
stay (1-4). Large retrospective studies in different countries
have demonstrated that 3% to 50% of patients experience

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

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Adverse events in elderly hospitalized patients
Szlejf C et al.

CLINICS 2012;67(11):1247-1252

Studies to detect AEs vary in the methodologies that are
used. Prospective observational studies have advantages
over retrospective studies for estimating AEs because they
can determine more events, particularly preventable ones,
and are more reliable (21,23,24). Most studies evaluate AEs
relative to all levels of care that are provided to the patients
(e.g., nurse and physician care and system-related factors)
without a thorough analysis of each specific level.
The aim of this prospective study was to determine the
frequency of medical AEs in the admissions of elderly
patients to an acute care geriatric ward and identify the
predictive factors of AEs and the correlation between AEs
and hospital mortality.

and/or health problem), immobility (defined as the inability to
change position in bed without help) and postural instability
(defined as two or more falls in the previous year).
During the first 24 hours after admission, the Charlson
Comorbidity Index (27), the Simplified Acute Physiology
Score II (SAPS II) (28), and the Katz Index of Independency
in Activities of Daily Living (29) were applied to evaluate
comorbidities, illness severity and functional status, respectively. The validated scales that were used in the study were
routinely applied to all of the patients in the geriatric unit.
During the hospitalization period in the geriatric ward, the
patients were evaluated daily by the multidisciplinary
health team. The occurrence of infections, delirium and
the prescription of potentially inappropriate drugs for the
elderly (based on the Beers criteria) were observed. The
length of stay in the geriatric unit and in-hospital mortality
were recorded at the end of the hospitalization period. The
Burden of Illness Score for Elderly Persons (30), which is a
risk adjustment system for older individuals who are
hospitalized, was calculated for each patient.
We defined a medical adverse event as an unintended
injury or complication that resulted in disability and was
caused by physician management rather than the patient’s
underlying disease process. Disability was defined as
temporary or permanent impairment of physical or mental
function. Major events were considered to be those events
leading to an increased mortality risk. System-related events
and events related to nursing care were not considered in
the present study. Any event that did not show a clear
cause-and-effect relationship to medical management and
subsequent adverse clinical manifestation was not considered iatrogenic. Potentially harmful conditions were
excluded if they did not involve injury to the patient. An
intervention that resulted in many harmful outcomes was
considered as a single AE. All of the events experienced by a
patient were included. The AEs that occurred before the
patient’s admission to the geriatric unit were not considered, even if the patient was still suffering the consequences
of the event.
The medical AEs were reported and briefly described by
the observer. A commission, which included three experienced geriatricians who were not involved in the data
collection or patient care, was formed to evaluate the AEs
that were described by the observer in monthly meetings.

METHODS
Study design and subjects
This observational and prospective study included the
admission of patients aged 60 years and older who had a
minimum stay of 24 hours in the acute care ward of the
geriatric unit at the Hospital das Clı´nicas da Faculdade de
Medicina da Universidade de Sa˜o Paulo (HCFMUSP)
between April 2007 and June 2008.
The patients were admitted directly from the emergency
room, the intensive care unit, or were referred by a geriatric
outpatient unit, day care hospital, homecare, or other
specialty unit.
The acute care ward in the geriatric unit has 10 beds for
elderly patients who present with pathologies that do not
initially require surgery or admission to the intensive care
unit. This ward is in HCFMUSP, which is a quaternary
university teaching hospital with 2,200 beds located in Sa˜o
Paulo, the largest city in Brazil.
The patients were treated by a multidisciplinary health
team whose members have been trained in gerontology,
including geriatric physicians and residents, nurses, nutritionists, physical therapists, speech pathologists and audiologists, psychologists, occupational therapists, and social
workers.
The patients who refused participation in the study and
did not sign the informed consent were excluded. The study
was approved by the HCFMUSP Ethics Committee and was
in accordance with the Helsinki Declaration of 1975.

Procedures
Statistical analysis

The patient data were recorded by one of the authors
(CS), a geriatrician who trained in detecting AEs during a
geriatrics residency. This observer was not involved in
patient care.
The data were obtained from the daily ward rounds and
directly from patients or their caregivers using pre-defined
questionnaires. If necessary, the observer had full access to
the patients’ charts.
The patient gender, age, data source (e.g., the patient or a
caregiver), diagnosis of infection, and drugs currently in use
(particularly medications considered inappropriate by the Beers
criteria) (25) were reported upon admission to the geriatric
ward. The occurrence of the following common geriatric
syndromes were also assessed: dementia and depression
(according to the Diagnostic and Statistical Manual of Mental
Disorders 4th edition [DSM-IV] criteria), delirium (in accordance
with the Confusion Assessment Method) (26), sphincter
incontinency (defined as the involuntary loss of urine or feces
in quantity and frequency sufficient to characterize it as a social

The data were analyzed using Student’s T-test, the chisquared test and Mann-Whitney U-test for continuous
quantitative variables with normal distribution, categorical
variables and qualitative ordinal variables, respectively. The
significance level was set at 5%. A backward stepwise
logistic regression model was conducted, the variables with
p-values ,0.10 in the univariate analyses were used to
determine the predictors of in-hospital death. Odds ratios
(OR) with CIs of 95% were calculated. The Nagelkerke’s Rsquared was used to determine the proportion of variation
explained by the model. These analyses were performed
using the SPSS statistical software, version 14 (SPSS, Inc.,
Chicago, IL).

RESULTS
During the study period, there were 238 sequential admissions to the acute care ward of the geriatric unit. Of these, 47

1248

CLINICS 2012;67(11):1247-1252

Adverse events in elderly hospitalized patients
Szlejf C et al.

patients refused to sign the informed consent. A total of 171
admissions were enrolled in the study; 101 female (59.1%). The
mean age of the sample was 78.12¡9.27 years. The patient
characteristics are shown in Table 1.
In 94 admissions (55%), 187 medical AEs occurred during
hospitalization. In 47.9% of the AEs, more than one event
occurred, with an average of 2.01 events per admission.
There were 103 major events. Examples of the frequent
iatrogenic events are shown in Table 2.
Patient data, including the age, gender, data informant,
presence of infection at the time of admission, geriatric
syndromes, functional status, prognostic indexes, number of
drugs prescribed, length of hospital stay, and hospital
mortalities were analyzed for AE associations. The predictors of medical AEs during hospitalization in the
geriatric ward were not observed in this study. The hospital
stay length and in-hospital mortality rates were higher in
the admissions with AEs (Table 1).
A model using in-hospital death as an endpoint was
developed. The patient data informant, sphincter incontinency, immobility, diagnosis of infection at admission,
SAPS II and Katz scores at admission, and occurrence of a
medical AE were significantly related to death in a
univariate analysis (Table 3). In a logistic regression, the
SAPS II score (OR = 1.13, CI 95%, 1.07-1.20, p,0.001), Katz
score (OR = 1.47, CI 95%, 1.18-1.83, p = 0.001), and occurrence
of a medical AE (OR = 3.59, CI 95%, 1.55-8.30, p = 0.003) were
predictors of death during hospitalization in the geriatric
unit (Table 4). Nagelkerke’s R-squared statistic was 0.40.
The patients with major events were more likely to die
during hospitalization than the patients with minor AEs (31
[47.7%] versus 7 [22.6%], p = 0.019).

admitted with AEs. In-hospital deaths could be predicted
based on the severity of illness measured by SAPS II, poorer
functional status at admission, and the occurrence of an AE
in the geriatric ward.
Although the frequency of AEs in the geriatric unit was
high, it was in alignment with the rates for older patients
that have been reported in the literature (1,2,4,6,11,12,16-20).
Because of the nature of the prospective study design and
the importance of determining a higher rate of AEs for
quality improvement, we used a more inclusive AE
definition than the major retrospective studies. We included
injuries that did not necessarily result in prolonged hospital
stays, disability at discharge, or death. Because most studies
analyze AEs in general as opposed to medical adverse
events, a comparison of the results of this study with other
studies is difficult.
The prospective study design and the less restrictive
definition of AEs could account for the high rate of
complications. The study setting may be a contributing
factor in that AEs have been reported to be more prevalent
in teaching hospitals (7,9,31). The rate of AEs reported in
studies in Brazil for all age groups ranges from 8% to 69%
(32-34). In one retrospective and one prospective study from
the same geriatric ward, AEs were less frequently reported
than in the present study (43.7% and 25.9%, respectively)
(18,20), which could be explained by the restricted population selected, including only those admitted to acute care
instead of all of the geriatric ward patients. This group of
patients tends to present with more severe illnesses and
may be more susceptible to complications during hospitalization.
We did not find any risk factors that were associated with
the occurrence of AEs. Other studies have reported that
educational level, non-elective hospitalization, admission to
a teaching hospital, hospital admission sector, functional
status, severity of illness, associated comorbidities, level of
consciousness, and the number of drugs prescribed at
admission (7,10,11,13-18,20) were related to AEs during
the hospital stay. However, most of these studies were

DISCUSSION
The present study found that 55% of hospital admissions
in a geriatric acute care ward were associated with a
medical AE and no risk factors were associated with the
occurrence of AEs. The length of hospital stay and inhospital mortality rate were higher in the patients who were

An event can be classified in more than one category. For example, a central venous catheter infection is a nosocomial infection and a therapeutic
procedure-related event.
{
Infection acquired during hospital care 48 hours after admission.
{
Nonsurgical therapeutic procedures
1
Delirium that started during the hospital stay in the geriatric ward and was apparently unrelated to drugs, therapeutic or diagnostic procedures.

risk of death was 1.47 in patients with medical injuries,
although the increased mortality risk associated with any
medical injury disappeared after a logistic regression model
was applied (14). The severity of illness as measured by the
SAPS II and poor functional status based on the Katz score
were also predictors of death, which is consistent with
another study (35).
The present study had some limitations. Because the
study was only performed in one hospital, the results may
not be generalized.
The data regarding AEs are subjective and dependent on
the judgment of the observer. To minimize this bias, all of
the events were analyzed by a specialized commission, and
those not considered as medical AEs were discharged. To
prevent a misinterpretation of the events, the observer was
not involved in patient care. Geriatricians are trained to
avoid medical adverse events. Therefore, the number of
events could be higher in other contexts.
It is difficult to separate AEs as a cause of morbidity or as
an effect of the patientsâ&#x20AC;&#x2122; comorbidities in observational
studies using hospitalized patients. Another limitation is the
exclusion of data regarding system-related AEs and the
events that are related to nursing care, which decreases the
reliability of the study.
Future studies should include additional variables, such
as race, income, number of AEs and the occurrence of major
AEs, to test for potential correlations and the predictors of
mortality.
Patients are increasingly exposed to diagnostic and
therapeutic procedures because of advances in medical
practice, and iatrogenic illnesses must be considered as
essential issues, particularly in older patients. Given that
there is not a characteristic risk profile for patients who are
susceptible to medical AEs, hospitalization and the actions
of the health team are the most common reasons for
occurrence. This study demonstrates that every elderly
hospitalized patient is at risk for medical AEs. The
consequences of AEs in older patients are extremely serious,
which makes this subject relevant for further studies to
better understand the mechanisms that could protect older
in-patients from medical harm.

retrospective and several were conducted in general hospitals and included all age groups.
In contrast to another study (20) that was conducted in the
same geriatric ward and included elective and acute
admissions in the analysis, the results of this study did
not indicate that delirium, the number of drugs prescribed
at admission, and the presence of postural instability
predicted the occurrence of AEs. This result may be caused
by the inclusion of a more heterogeneous group of patients
in the previous study (acute and elective admissions).
Another possibility is that the definition of AEs was
restricted to medical adverse events in the present study.
The absence of predictors of medical AEs during the
hospitalization of elderly patients suggests the hypothesis
that the occurrence of these complications is not dependent
on the condition of the patient at the time of admission.
Although some events may be caused by latent errors and
are not related to medical care, physicians must carefully
assess their actions to protect patients from medical AEs.
Another important issue highlighted in this study is that
medical adverse events led to longer hospital stays and
were related to in-hospital deaths, which is similar to the
results of previous research (7,8,10,11,14,16,18,33). The
relationship between AEs and the length of hospital stay
is not clear because it is not possible to determine if a longer
hospital stay renders patients more susceptible to complications or if the AEs prolong the hospitalization.
A primary contribution of this study is that medical AEs
are independent predictors of in-hospital death, even after
adjusting for confounding factors. In a study performed in
acute care hospitals, Meurer et al. found that the relative

AUTHOR CONTRIBUTIONS
Szlejf C provided substantial contributions to the conception and design of
the study, data acquisition, analysis and interpretation, drafting of the
manuscript, and final approval of the manuscript. Farfel JM, Curiati JA
and Jacob-Filho W contributed to the conception and design of the study
and final approval of the manuscript. Couto Junior EB contributed to the
analysis and interpretation of data. Azevedo RS contributed to the
conception and design of the study, analysis and interpretation of data,
critical revision of the article and final approval of the manuscript.

The role of the resistive index in Hashimoto’s thyroiditis: a Sonographic pilot study in children
Basar Sarikaya,I,III Huseyin Demirbilek,II Deniz Akata,I Nurgun KandemirII
I
Hacettepe University Medical Faculty Department of Radiology, Ankara, Turkey. II Hacettepe University Medical Faculty, Department of Pediatrics,
Ankara, Turkey. III University of Minnesota, Department of Radiology, Minneapolis/MN, USA.

OBJECTIVE: The role of Doppler ultrasonography in the diagnosis of diffuse thyroid diseases is not well established.
In particular, Doppler ultrasonography findings in children with Hashimoto’s thyroiditis are very limited. We
examined gray-scale and Doppler ultrasound findings in Hashimoto’s thyroiditis in children in an attempt to
understand the feasibility of future prospective controlled studies.
MATERIALS AND METHODS: Twenty-one children with newly diagnosed Hashimoto’s thyroiditis were recruited in
the study. The patients were euthyroid or had subclinical hypothyroidism at the time of the ultrasonography
examination. According to the color Doppler scale developed by Schulz et al., thyroid glands were classified into
four patterns based on visual scoring and the mean resistive index (RI), which was calculated via measurements from
both lobes, and these results were compared with gray-scale findings.
RESULTS: The mean RI value, calculated as the mean of the RI values of both lobes obtained from each patient, was
found to be 0.57¡0.05 (range 0.48-0.67) cm/sn. The distribution of thyroid classifications was as follows: Pattern 0,
n = 7; Pattern I, n = 6; Pattern II, n = 4; and Pattern III (‘‘thyroid inferno’’), n = 4. The mean RI values in patients with
normal or near-normal gray-scale findings (n = 10) and patients with more substantial gray-scale changes (n = 11)
were not significantly different and were lower than the values in normal children previously presented in the
literature.
CONCLUSION: The results indicated that the RI may be more sensitive than other ultrasound parameters for the
diagnosis of Hashimoto’s thyroiditis.
KEYWORDS: Doppler Ultrasound; Hashimoto’s Thyroiditis.
Sarikaya B, Demirbilek H, Akata D, Kandemir N. The role of the resistive index in Hashimoto’s thyroiditis: a Sonographic pilot study in children. Clinics.
2012;67(11):1253-1257.
Received for publication on June 10, 2012; First review completed on July 10, 2012; Accepted for publication on July 10, 2012
E-mail: basarsarikayamd@yahoo.com
Tel.: 612 626-7741

a color Doppler US pattern in Graves’ disease that was not
observed in normal individuals or in patients with other
thyroid diseases and named it the ‘‘thyroid inferno’’. This
pattern results from continuous multiple intrathyroidal
flows during systole and diastole (15).
However, to the best of our knowledge, no previous study
has described Doppler US findings in children with
Hashimoto’s disease. In this preliminary study, we aimed
to compare conventional ultrasonography (gray scale) with
color Doppler US findings in newly diagnosed patients with
Hashimoto’s disease.

INTRODUCTION
Ultrasonography has been used in the diagnosis of diffuse
thyroid diseases for many years (1-10). The diagnostic role
of Doppler ultrasonography (US) in diseases of the thyroid
was evaluated in studies on thyroid nodules during the
1980s and 1990s (11,12). However, the use of color Doppler
US in thyroid disease is a relatively new and promising
concept. Previously published studies mainly focused on
the detection of adenomas and the differentiation of
adenomas from carcinomas in cases of cold nodules with
different points of view (13,14).
Limited information on the role of color Doppler US in
diagnosing diffuse thyroid diseases, such as Hashimoto’s
disease, exists in the literature. Ralls et al. initially described

MATERIALS AND METHODS
This study was conducted by a retrospective review of
digitally stored US images of newly diagnosed Hashimoto’s
thyroiditis patients. The subject population consisted of a
small portion of a larger cohort of patients referred to the
Ultrasound Unit from the Pediatric Endocrinology Unit of
Hacettepe University Medical Center, Ankara, Turkey (16).
Twenty-one patients were included over an 18-month
period, including 19 females and two males, with ages

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

Diffusely enlarged gland with a normoechoic (similar to normal tissue) pattern
Multiple hypoechoic foci or patches scattered throughout an otherwise normoechoic gland; a pattern
suggestive of focal rather than diffuse involvement
Enlarged gland with diffuse but mild hypoechogenicity
Enlarged gland with diffuse and marked hypoechogenicity

ranging from 6-12 years (median age = 10 years). The study
was approved by the Hacettepe University Medical School
Institutional Review Board, and informed consent was
waived. Inclusion criteria included a new diagnosis of the
disease with no history of treatment and euthyroid or
subclinical hypothyroidism. Doppler US was performed as
an adjunct to routine clinical sonographic evaluation of the
patients.
Each subject underwent ultrasound examination using a
Sonoline Elegra (Siemens, Erlangen, Germany) sonographic
machine with a 7.5-MHz transducer. Ultrasound examinations were performed by the same researcher for all
patients. Gray-scale ultrasonography parameters included
echogenicity and size of the thyroid gland. The total thyroid
volume in each patient was compared with upper level
values determined for specific age groups by the World
Health Organization (WHO), and patients with thyroids
larger than the reference values were classified as having
thyromegaly (17). The echogenicity of each individual
thyroid gland was noted and used in the classification of
gray-scale findings based on a classification system originally created by Sostre and Reyes (Table 1) (4). However, a
modification to the original classification was required to
account for patients with completely normal gray-scale
findings (Grade 0).
Color Doppler US examination was performed by setting
the pulse repetition frequency (PRF) and color Doppler gain
to appropriate levels (the maximum gain and minimum PRF
at which no aliasing in the carotid artery or internal jugular
vein was observed) in all patients. The vascularity of both
lobes was determined based on a visual scale according to
the classification previously created by Schulz et al. (Table 2)
(18).
RI measurements were performed within each lobe of the
thyroid at a location close to the center, where vascularity
could still be observed. The values obtained for each lobe
were averaged for each patient, and a mean RI value for the
entire patient group was calculated. Mean RI values were
also calculated for each Doppler pattern. The RI values
of patients with normal gray-scale findings or minimal
changes (Grade 0 or 1) were compared with those of
patients with strongly positive gray-scale findings (n = 11).

RESULTS
The patients’ laboratory findings were as follows. Nineteen
patients had positive thyroid antibodies (one or more of the
following antibodies: antithyroglobuline (ATA), anti-thyroid
peroxidase (anti-TPO), and anti-microsomal (AMA) antibodies). Two patients had negative values at the time of US
examination but had previously tested positive for thyroid
antibodies. Sixteen patients were euthyroid during US examination, and five patients had subclinical hypothyroidism.
The thyroid gland volume was within normal limits
(according to the WHO reference) in 13 patients, and eight
patients had thyromegaly. According to the modified Sostre
and Reyes gray-scale classification, thyroid glands were
scored as Grade 0 in seven patients, Grade 1 in three
patients, Grade 2 in seven patients, Grade 3 in two patients
and Grade 4 in two patients (Table 3; also see Figure 1).
According to the color Doppler scale of Schulz et al. (17),
seven patients had Pattern 0, six patients had Pattern I, five
patients had Pattern II, and four patients had Pattern III
(‘‘thyroid inferno’’) thyroid glands (Figure 2 and Table 4).
The mean RI value, calculated as the mean of the mean RI
values of both lobes in all patients, was 0.57¡0.05 (range
0.48-0.67) cm/sn.
Thyroid glands that were classified as having normal or
near-normal gray-scale findings (n = 10) and those with
substantial gray-scale changes (n = 11) demonstrated no
statistically significant difference in the RI. Patients with
minimal or no US gray-scale findings were found to have a
mean RI value of 0.58¡0.056 cm/sn, and patients with
substantial gray-scale changes had a mean RI value of
0.56¡0.059 cm/sn.
In addition, the mean RIs calculated for each Doppler
pattern were found to range from 0.56 to 0.58 (Table 5).

DISCUSSION
Hashimoto’s thyroiditis is the most common cause of
goiter and hypothyroidism in children (19-21). Nearly all
cases of thyroiditis seen in children are cases of Hashimoto’s
disease (21). In the diagnosis of Hashimoto’s thyroiditis, two
different diagnostic criteria exist, namely one developed by

Table 3 - Grading of the gray-scale findings and
distribution of patients within the grades. (Note: we have
added Grade 0 to the original grading system created by
Sostre and Reyes).

muscles. Because the thyroid gland is expected to be more
echogenic than adjacent muscles in normal individuals, the
authors emphasized that a hypoechoic gland may be
suggestive of hypothyroidism and that ultrasonography
may be an important easy and noninvasive method for
diagnosing Hashimoto’s disease (2).
In the absence of an ideal test for Hashimoto’s thyroiditis,
Sostre and Reyes proposed that thyroid US could be an
appropriate diagnostic test. They used a grading system to
classify ultrasonographic patterns into four groups, with the
sternomastoid muscle chosen as a reference (4). However,
because Sostre and Reyes’ classification is based solely on
the presence of ultrasonographic changes, there is no
group in the classification that corresponds to clinical and
laboratory findings of Hashimoto’s disease or to findings
of completely normal ultrasonographic examinations.
Therefore, in this study, it was deemed appropriate to add
Grade 0 to their original grading system. In the present
study, we found that a high grade was correlated with
thyroid gland destruction and hypothyroidism. Five
patients with subclinical hypothyroidism were found to
have Grade 3 or Grade 4 disease.
Bogazzi et al. investigated the cause of the increase in
thyroid blood flow in untreated Graves’ disease patients,

Fischer et al. and the other developed by the Japan Thyroid
Association. These two criteria, while different, are both
based on clinical and laboratory findings (22). Imaging of
the thyroid gland (ultrasonography and scintigraphy) is not
included in the diagnostic criteria because of its low
specificity. The most objective finding in thyroid ultrasonography is the quantitative measurement of thyroid volume.
The WHO has defined the standard normal upper limits of
ultrasonographically measured thyroid volumes according
to age and gender because only 50% of goiter classifications
can be accurately made by palpation. In an article published
in 1999, the authors evaluated intraobserver and interobserver differences and found no significant differences,
therefore concluding that thyroid ultrasonography was
reliable for evaluating thyroid dimensions and volume (23).
US is valuable for determining the presence of nodular
goiter in patients with Hashimoto’s disease and can enable
the characterization and surveillance of these nodules.
Furthermore, US also helps to position and guide fineneedle aspiration biopsy (24,25).
Hayashi et al. investigated ultrasound findings in 53
histologically confirmed patients with diffuse thyroid diseases and classified the thyroid echogenity into groups A and
B, as iso-, hypo-, or hyperechoic compared with adjacent

compared 20 children with normal thyroids, with 20
children with endemic goiter and found that RI values
were significantly lower in the disease group (32). The mean
RI was 0.58¡0.05 in the disease group and 0.70¡0.05 in the
control group. Although the measurements were taken from
thyroid arteries, rather than from the gland itself, the RI
value in the disease group was in accordance with the mean
RI value in our study (0.57¡0.05).
The major conclusion of our study is that, despite
variations in color Doppler patterns, there was no statistically significant difference in the mean RI values between
patients with normal or near-normal gray-scale findings
and patients with marked gray-scale changes. Both RI
values were below normal limits. This finding indicates that
it is important to evaluate the RI even in subjects with
sonographically normal thyroid glands.
Conversely, subjective color Doppler grading of the
thyroid gland did not yield the expected results. Specifically, a small group of patients (38%, 8/21) was found to
have markedly increased vascularity of the thyroid.
This study has several limitations. The major limitation is
the lack of a control group. In future studies designed to test
the efficacy of RI, the presence of a control group of age- and
gender-matched subjects free of thyroid disease is crucial.
Another limitation is the small size of the patient group,
which limited our ability to perform any statistical analysis.
Of the different spectral Doppler US parameters, only the RI
was tested in this study. Other parameters, such as the peak
systolic velocity, end diastolic velocity and pulsatility index,
could be examined in future studies. The lack of assessment
of the interobserver and intraobserver variability, due to the
retrospective nature of the study, is another important
limitation. This particular issue is extremely important for
studies based on US imaging, a method known to result in
significant interobserver and intraobserver variability.
Color Doppler imaging in Hashimoto’s disease appears to
be a promising diagnostic imaging modality. In particular,
the changes in RI values in patients with relatively normal
gray-scale findings prompt us to suggest adding Color
Doppler imaging to routine ultrasound examination of those
patients. Further blinded, controlled studies with a sufficient
number of patients are required to determine measures of test
performance of RI in Hashimoto’s disease and would aid in
determining the cut-off point for a normal RI value.

and they observed thyroid vascularity in different subgroups corresponding to different thyroid disease types
(26). These authors stated that thyroid hormones were not
the cause of increased thyroid vascularity but that TSHreceptor antibodies or TSH may be the cause. This
hypothesis was based on the fact that intrathyroidal
vascularity and flow velocity increase in spontaneous
hyperthyroidism but not in hyperthyroidism secondary to
thyroid hormone intake or thyroid gland destruction. In
addition, an increase in vascularity and flow velocity is also
seen in Hashimoto’s disease patients with hypothyroidism
(26). In the same study, intrathyroidal peak systolic flow
velocity was thought to be a better index of thyroid disease
because it demonstrated a more significant increase in
patients with Graves’ disease than in those with
Hashimoto’s disease. Iitaca et al. examined vascular
endothelial growth factor (VEGF), which is an antigenic
growth factor, and concluded that a significant relationship
existed between intrathyroidal flows and VEGF levels (27).
Once it was realized that hypervascularity was not unique
to hyperthyroidism, Caruso et al. evaluated flow velocity in
autoimmune thyroid diseases and concluded that the
inferior thyroid arterial peak systolic flow velocity exceeded
150 cm/sec in patients with these diseases. However, the
velocity remained within normal limits in patients with
other thyroid diseases and did not exceed 65 cm/sec. These
authors emphasized the importance of inferior thyroid
artery peak systolic flow velocity in the differential
diagnosis of diffuse thyroid disease and follow-up care of
patients with Graves’ disease (28).
Schulz et al. investigated the role of color Doppler US in
hypothyroidism and, in reference to previous studies,
classified the vascularity (Table 2) (18). In their study, it
was reported that the hypervascularity found in patients
with Graves’ disease was also present in patients with
hypothyroidism to some extent.
There is no consensus regarding normal values for
Doppler parameters measured in the thyroid gland or
inferior thyroid artery, namely the resistivity index (RI),
pulsatility index (PI) and peak systolic flow velocities. In
addition, no common guidelines exist on how to obtain
these parameters (29-31). Mahmutyazicioglu and Turgut

OBJECTIVE: Pleural tuberculosis is the most frequently occurring form of extra pulmonary disease in adults. In up to 40%
of cases, the lung parenchyma is concomitantly involved, which can have an epidemiological impact. This study aims to
evaluate the pleural and systemic inflammatory response of patients with pleural or pleuropulmonary tuberculosis.
METHODS: A prospective study of 39 patients with confirmed pleural tuberculosis. After thoracentesis, a high resolution
chest tomography was performed to evaluate the pulmonary involvement. Of the 39 patients, 20 exhibited only pleural
effusion, and high resolution chest tomography revealed active associated-pulmonary disease in 19 patients. The total
protein, lactic dehydrogenase, adenosine deaminase, vascular endothelial growth factor, interleukin-8, tumor necrosis
factor-a, and transforming growth factor-b1 levels were quantified in the patient serum and pleural fluid.
RESULTS: All of the effusions were exudates with high levels of adenosine deaminase. The levels of vascular
endothelial growth factor and transforming growth factor-b1 were increased in the blood and pleural fluid of all of
the patients with pleural tuberculosis, with no differences between the two forms of tuberculosis. The tumor
necrosis factor-a levels were significantly higher in the pleural fluid of the patients with the pleuropulmonary form
of tuberculosis. The interleukin-8 levels were high in the pleural fluid of all of the patients, without any differences
between the forms of tuberculosis.
CONCLUSION: Tumor necrosis factor-a was the single cytokine that significantly increased in the pleural fluid of the
patients with pulmonary involvement. However, an overlap in the results does not permit us to suggest that
cytokine is a biological marker of concomitant parenchymal involvement. Although high resolution chest
tomography can be useful in identifying these patients, the investigation of fast acid bacilli and cultures for M.
tuberculosis in the sputum is recommended for all patients who are diagnosed with pleural tuberculosis.
KEYWORDS: Cytokines; Inflammation; Pleural Diseases; Tuberculosis.
Antonangelo L, Vargas FS, Puka J, Seiscento M, Acencio MM, Teixeira LR, Terra RM, Sales RK. Pleural tuberculosis: is radiological evidence of
pulmonary-associated disease related to the exacerbation of the inflammatory response? Clinics. 2012;67(11):1259-1263.
Received for publication on April 25, 2012; First review completed on May 13, 2012; Accepted for publication on July 12, 2012
E-mail: roberta.sales@uol.com.br
Tel.: 55 11 2661-5695

accepted that pleural tuberculosis results from a late
hypersensitive reaction to the antigens of M. tuberculosis
subsequent to the rupture of a subpleural caseous focus
(1,2,4). The release of even a small number of bacilli from the
lungs to the pleural space triggers a series of immune
reactions that are mediated by T lymphocytes, which
produce cytokines and stimulate macrophages to form a
granuloma (4-7). These events trigger an inflammatory
process in the pleural cavity: vascular permeability
increases and an influx of leukocytes enter the pleural
space, resulting in the accumulation of fluid and cells,
which is a characteristic of the pleural exudates (1,2,6).
Cytokines are a group of polypeptides with multiple
biological functions that act with other inflammatory
mediators to coordinate the interaction between sensitized

INTRODUCTION
Pleural tuberculosis is the most common extra pulmonary
form of tuberculosis in adults, particularly in countries
where the prevalence of the disease is high or moderate (13). Although pulmonary tuberculosis can be a manifestation
of primary infection, it is most commonly associated with
the reactivation of pre-existing foci (1,2,4). It is generally

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

1259

Pleural inflammation in tuberculosis
Antonangelo L et al.

CLINICS 2012;67(11):1259-1263

bacilloscopy, M. tuberculosis cultures, and to determine the
adenosine deaminase levels (Giusti modified method) (26).
Proteins (Biuret method) and lactic dehydrogenase (enzymatic method) were also quantified in the blood and pleural
fluid. Aliquots from both samples (serum and pleural fluid)
were immediately centrifuged (1,500 rpm for 10 minutes at
4 ˚C), and the supernatant was stored at -80 ˚C for posterior
cytokine analyses.
VEGF, IL-8, TNF-a, and TGF-b1 were quantified using an
immunoenzymatic method (enzyme-linked immunosorbent
assay, ELISA) according to the manufacturer’s instructions
(R&D Systems Inc., Minneapolis, USA). The assays were
performed in triplicate, and the results are expressed as
means. The results were quantified by comparing the optic
density (450 nm filter) in the ELISA reader (Powerwave,
Biotek, USA) against a pre-established standard curve. The
minimal detection values for IL-8, VEGF, and TGF-b1 were
31 pg/mL, and the minimum detection value for TNF-a was
16 pg/mL. To evaluate the pleural inflammatory response,
the results were compared with those obtained from the
serum and pleural fluid of the patients with transudates that
were caused by heart failure (27).

lymphocytes and macrophages to form granulomas (5-11).
Among the involved mediators, we highlight the importance of interleukin-8 (IL-8) (8-15), vascular endothelium
growth factor (VEGF) (8,9,16-18), tumor necrosis factor-a
(TNF-a) (8,9,11,17-20), and transforming growth factor-b1
(TGF-b1) (7,9,20,21), all of which are considered to be key
effectors in the inflammatory response of the pleural space
(4-6).
Although pleural and pulmonary lesions were previously
considered to be independent events, the advent of computed
tomography has demonstrated concomitant lesions in more
than 40% of cases (1,21-24). This concomitance may have an
epidemiological impact, given that many patients with
pleural tuberculosis are not adequately evaluated for intrathoracic involvement. This finding enables us to speculate
whether serum or pleural fluid inflammatory markers in
pleural tuberculosis could suggest associated active pulmonary disease. The answer to this question is particularly
relevant if we consider the potential risk of Mycobacterium
tuberculosis transmission from these patients to contacts.
Therefore, this study compares the serum and pleural
fluid expression of inflammatory mediators in pleural
tuberculosis patients to evaluate whether pulmonary-associated involvement influences the magnitude of the inflammatory response. We hypothesized that the more extensive
the injury, the greater the inflammatory response. If this
hypothesis is true, the inflammatory cytokines could be
increased both locally (pleural fluid) and in the serum as a
result of the pulmonary involvement.

Statistical analysis
The results of the inflammatory marker analysis were
compared using Student’s T-test or the Mann Whitney Utest according to the distribution of the variables. The
SigmaStat 3.5 (SSI 2006, California, USA) program was used
for the statistical analyses, and the results are presented as
medians (IQ 25th – 75th); p#0.05 was considered to be
statistically significant.

METHODS
After approval by the local ethics committee, informed
consent was obtained from 39 pleural tuberculosis patients.
The patients were prospectively selected from the outpatient clinic of Pulmonary Diseases (InCor/FMUSP), Sa˜o
Paulo, Brazil.
The diagnosis of pleural tuberculosis was based on the
presence of a granuloma (upon pleural biopsy) associated
with exudative effusion with increased adenosine deaminase
(ADA . 40 IU/L) and/or a culture of the pleural fluid or
fragment positive for Mycobacterium tuberculosis (1,2,25). After
thoracentesis, the patients underwent high resolution chest
tomography (HRCT) and were subdivided into two groups
based on the HRCT findings: pleuropulmonary (n = 19) or
only pleural involvement (n = 20). The tomographic abnormalities that were suggestive of active pulmonary disease
included the following: multi-segmental consolidation over
the upper lung zones (homogenous opacity that reflected
granulomatous inflammation of the parenchyma), thickwalled cavities (resulting from the coalescence of multiple
inflammatory foci that necrotized and drained into the
airways), centrilobular or confluent nodules, and the presence
of the tree-in-bud pattern, which reflects the endobronchial
dissemination of caseous necrosis and the granulomatous
inflammation that fills and surrounds the alveolar ducts and
respiratory bronchioli. Patients who had nonspecific tomographic findings or images of residual scarring were not
included in this series (23-25). HIV patients, patients
previously treated for tuberculosis, and patients undergoing
immunosuppressant therapy were also excluded.
Concomitant with thoracentesis, all of the patients underwent peripheral venous puncture and tuberculin skin
tests. Pleural fluid samples were processed for cytology,

RESULTS
Thirty-nine patients (35¡15 years) presented with pleural
effusions caused by tuberculosis, and 19 patients exhibited
concomitant parenchyma involvement. In the patients with
pleural effusion (n = 20), the detection of fast acid bacilli in
the sputum was negative. In the pleuropulmonary group
(n = 19), three patients exhibited positive fast acid bacilli in
sputum. In two patients in the pleural group and six
patients in the pleuropulmonary group, the response to the
skin test exhibited indurations that were greater than 5 mm.
Thus, the microbiological detection and the skin test
contributed little toward diagnosing active pulmonary
tuberculosis. The patients were not submitted to bronchoalveolar lavage because they had previously been diagnosed with pleural tuberculosis.
The HRCT findings that were considered to be suggestive
of active pulmonary disease met the following criteria: the
presence of centrilobular nodules (n = 14), confluent nodules
(n = 11), tree-in-bud pattern (n = 15), pulmonary consolidation in the upper lung zones (n = 8), and thick-walled
cavities (n = 3). Some patients exhibited more than one of
these radiological changes.
Tuberculous pleural effusions were exudates with high
ADA levels according to Light’s criteria. Comparing with
transudates, the levels of all inflammatory markers were
higher in the pleural fluid of the tuberculosis patients (data
not shown). VEGF and TGF-b1 were increased to similar
levels in the blood and pleural fluid of the patients with
both forms of the disease. The TNF-a levels were significantly higher in the pleural fluid of the patients with the
pleuropulmonary form of the disease; this difference

represented the single parameter that was capable of
differentiating between the two forms. IL-8 was increased
in the pleural fluid of all the TB patients, and no differences
in the levels of IL-8 were observed between the two forms.
As expected, the protein and lactic dehydrogenase levels
were higher in the serum and pleural fluid, respectively
(Table 1; Figures 1 and 2).

896 (288–1781)
31 (31–125)
16 (16–475)
751 (486–1135)

0.014
,0.001
0.007
0.439

DISCUSSION
The high expression of inflammatory mediators in the
pleural fluid of pleural tuberculosis patients allows us to
recognize the exudative inflammatory response at the site of
active disease. The increased levels of VEGF and TGF-b in
the serum of these patients reflect the systemic response

Figure 1 - Biochemical analysis of the fluid and blood of the patients with tuberculous pleural effusions. The data are expressed as
medians: 25th and 75th percentiles (*p,0.05: fluid x blood).

observed in cases of pleural tuberculosis. Although elevated
TNF-a levels in the pleural fluid was the single parameter
that was capable of differentiating between the two forms of
the disease, the overlapping results do not suggest that this
cytokine is a marker of pulmonary involvement.
Tuberculosis pleurisy is an acute and symptomatic
disease that invariably evolves with exudative pleural
effusions that are rich in cells and inflammatory mediators
(1,2,4,7-10). Although tuberculosis pleurisy can indicate a
manifestation of primary infection, it is most commonly
associated with the reactivation of preexisting foci (1,2,4).
Currently available laboratory methods frequently fail to
demonstrate a possible concomitance with active pulmonary disease (1). In this regard, high resolution chest
tomography has been helpful in identifying pulmonary
lesions that are consistent with active disease in approximately 40% of pleural tuberculosis patients (20-24). This
finding has epidemiological implications, particularly if we
consider that these patients are potential sources of infection
and that their contacts are often not clinically or radiologically evaluated. Therefore, we hypothesized that the
patients with the pleuropulmonary form of tuberculosis
could exhibit a greater inflammatory response because of
the involvement of more than one anatomic site. This fact
presumably determines systemic repercussions that could
be detected by evaluating the serum and pleural inflammatory mediators.

Our findings corroborate previously described reports of
increased inflammatory mediators in the pleural fluid of
patients with pleural tuberculosis, thereby providing evidence of the compartmentalization of the inflammatory
response at the active disease site (10,13,20,27). During the
early phases of infection, after antigenic stimulation is
triggered by the mycobacteria, cytokines such as VEGF,
TNF-a, and IL-8 are transiently produced in vivo and alter
the pleura permeability; this event precedes the exudative
phase (20,28). However, the elevated serum levels of TNF-a
and TGF-b are thought to be related to prolonged antigenic
stimulation, similar to observations in patients with pulmonary tuberculosis or late-diagnosed pleural tuberculosis
(28-30).
In pleural tuberculosis, many cytokines are produced
intracavitarially and modulate the inflammatory response
that is triggered by the mycobacteria or its antigens to limit
or aggravate the disease (7,19). Previous studies have
related the findings of high serum or PF levels of TNF-a,
TGF-b, and IL-8 to residual pleural thickening (19,20). TGFb is a mediator that is expressed in active infection and plays
a fundamental role in the fibrotic scaring process. TGF-b is a
key mediator in the immunopathogenesis of tuberculosis
because it is able to modify the production and function of
other cytokines, such as IL-1b and TNF-a, in addition to
modulating the functions of T lymphocytes and macrophages (7,20,29,30). In pleural tuberculosis, the excessive

1262

CLINICS 2012;67(11):1259-1263

Pleural inflammation in tuberculosis
Antonangelo L et al.

production of TGF-b is believed to be related to the clinical
progression of the disease, particularly in the physiopathology of pleural thickening (29). TGF-b possesses proinflammatory activity in low concentrations (pleural tuberculosis
and healthy contacts of tuberculosis carries) and antiinflammatory activity in high concentrations (pulmonary
tuberculosis). We observed increased levels of TGF-b in the
pleural fluid and blood of tuberculosis patients. Although
higher TGF-b levels were observed in the pleuropulmonary
form, there was no statistical significance when compared to
the levels in patients with pleural disease.
Siawaya et al. (8) studied the cytokine and chemokine
profiles of patients with different forms of tuberculosis to
better understand the immunopathology of the disease and
identify the biological markers that differentiate the various
clinical forms of the disease. The authors concluded that
systemic inflammatory markers, such as IL-8, TNF-a, and
VEGF, are associated with pleural tuberculosis, whereas
elevated levels of the factors involved in cell-mediated
immunity, such as IL-12p40 and sCD40L, characterize
pulmonary tuberculosis.
In conclusion, the present study demonstrates an intense
intracavitary inflammatory response in the patients with
pleural tuberculosis independent of parenchymal involvement. Although all of the cytokines were overexpressed in
the pleural fluid, only TNF-a was significantly increased in
the pleuropulmonary tuberculosis patients. This finding
could suggest a more pronounced inflammatory response
because of the concomitance of the anatomic sites involved.
However, TNF-a should not be considered to be a biological
marker of pulmonary-associated disease. Although HRCT
can be useful in identifying these patients, the identification
of fast acid bacilli and/or the culture for M. tuberculosis in the
sputum is recommended for all pleural tuberculosis patients.

9.
10.

11.

12.

13.

14.
15.

16.
17.

18.

19.
20.

ACKNOWLEDGMENTS

21.

The study was supported by grants from the Fundac¸a˜o de Amparo a`
Pesquisa do Estado de Sa˜o Paulo (FAPESP).

OBJECTIVE: The standard therapy for patients with high-level spinal cord injury is long-term mechanical ventilation
through a tracheostomy. However, in some cases, this approach results in death or disability. The aim of this study is
to highlight the anesthetics and perioperative aspects of patients undergoing insertion of a diaphragmatic
pacemaker.
METHODS: Five patients with quadriplegia following high cervical traumatic spinal cord injury and ventilatordependent chronic respiratory failure were implanted with a laparoscopic diaphragmatic pacemaker after
preoperative assessments of their phrenic nerve function and diaphragm contractility through transcutaneous nerve
stimulation. ClinicalTrials.gov: NCT01385384.
RESULTS: The diaphragmatic pacemaker placement was successful in all of the patients. Two patients presented
with capnothorax during the perioperative period, which resolved without consequences. After six months, three
patients achieved continuous use of the diaphragm pacing system, and one patient could be removed from
mechanical ventilation for more than 4 hours per day.
CONCLUSIONS: The implantation of a diaphragmatic phrenic system is a new and safe technique with potential to
improve the quality of life of patients who are dependent on mechanical ventilation because of spinal cord injuries.
Appropriate indication and adequate perioperative care are fundamental to achieving better results.
KEYWORDS: Spinal Cord Injury; Quadriplegia; Pacemaker; Artificial Diaphragm; Anesthetic; Perioperative
Management.
Tedde ML, Vasconcelos Filho P, Hajjar LA, Almeida JP, Flora GF, Okumura EM, et al. Diaphragmatic pacing stimulation in spinal cord injury: anesthetic
and perioperative management. Clinics. 2012;67(11):1265-1269.
Received for publication on May 16, 2012; First review completed on June 21, 2012; Accepted for publication on July 16, 2012
E-mail: tedde@usp.br
Tel.: 55 11 2661 5708

adverse effects, such as a higher incidence of lung infection
and death (2). Furthermore, in USA, the estimated life
expectancy for a 20-year-old patient who has an SCI and is
dependent on mechanical ventilation decreases from 58.6
to 17.1 years (1).
The concept of phrenic nerve stimulation, or electric
ventilation, is not new (3). More recently, a new device that
focuses on the phrenic nerve motor point stimulation on the
abdominal portion of the diaphragm was developed to
allow patients to be weaned from mechanical ventilation (2).
The inclusion criteria for diaphragmatic pacemaker implantation are chronic ventilator-dependent high-level SCI, a
stimulable diaphragm and preserved phrenic nerves. A
successful result depends on the ability of the pacemaker
system to provide adequate tidal volume and to ultimately
allow weaning from the mechanical ventilation (4,5).
There are few previous publications concerning the perioperative management of patients undergoing diaphragmatic

INTRODUCTION
Spinal cord injury (SCI) is a serious condition that mainly
affects young adults and often results in death or disability.
The critical loss of neurological function below the level of
the injury leads to multiple adverse effects, particularly in
the respiratory system. Approximately 50% of SCI patients
develop quadriplegia, with 4% requiring mechanical
ventilation (1). The standard therapy for patients with
high-level SCI is long-term mechanical ventilation through
tracheostomy. However, this treatment is associated with

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

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CLINICS 2012;67(11):1265-1269

pressure [SAP] lower than 90 mmHg or mean arterial
pressure [MAP] lower than 65 mmHg) during peritoneal
insufflation, a 500 mL bolus of lactated Ringer’s was
administered. If hypotension persisted, 5 mg ephedrine in
bolus was administered. None of our five patients presented
with dysreflexia or vasopressor/inotropic support requirements.
The initial phase included mapping each hemidiaphragm
by systematically stimulating it under direct vision to
determine two sites for the permanent pacing of the phrenic
nerves (Figure 1). These permanent electrodes were inserted
into the diaphragm: one centrally and one posteriorly
(Figure 2). A clinical station (Synapse Biomedical, Oberlin,
OH, USA) was used to establish the parameters of the
pacing device (Figure 3).
After implantation, the DPS was tested by switching the
ventilation mode of the anesthesia machine to the spontaneous mode. An electrocardiogram strip was also recorded
to ensure that the left-sided pacing did not affect the cardiac
rhythm (8).

pacemaker implantation. The aims of this study are to
report on five cases of high cervical SCI treated with the
laparoscopic insertion of a diaphragmatic pacemaker and
to highlight the anesthetic and perioperative aspects of
each case.

MATERIALS AND METHODS
The patients in this report were part of a funded pilot
project conducted by the thoracic surgery and neurosurgery
departments at the Heart Institute (InCor, Sa˜o Paulo, SP,
Brazil) (6). This study was approved by the InCor Ethical
Committee (CAPPesq n 0551/10).
Five patients presenting with quadriplegia after high
cervical traumatic SCI and ventilator-dependent chronic
respiratory failure were scheduled to undergo laparoscopic
implantation of the NeuRxH Diaphragm Pacing System
(DPS) (Synapse Biomedical, Oberlin, OH, USA).
All of the patients were selected after preoperative
assessments of their phrenic nerve function and diaphragm
contractility using transcutaneous nerve stimulation. All of
the patients were tracheostomized and dependent on
mechanical ventilation. The patients’ clinical and demographic data were recorded (Table 1).
Preoperative anesthetic assessments were performed, and
all of the patients were pre-medicated with 0.1 mg/kg oral
midazolam 30 minutes before arriving in the operating
room. The induction of anesthesia was achieved with
intravenous 5 mg/kg fentanyl and 2 mg/kg propofol. The
bi-spectral index (BIS) was used during anesthesia, and a
range of 40–60 was maintained during the procedure to
allow for adequate sedation levels. No neuromuscular
blockers were given during the procedure. Anesthesia was
maintained with 1.5–2% sevoflurane and a mixture of 60%
oxygen and nitrogen; 1 mg/kg fentanyl was administered in
bolus according to anesthetic criteria. If appropriate
hypnosis was not reached, an intravenous infusion of 0.25
mg/kg/min propofol was started. The patients were
monitored with electrocardiography, pulse oximetry, capnometry, and non-invasive arterial pressure. All of the
patients were ventilated with a tidal volume of 8 ml/kg.
The respiratory rate was maintained at 10–16 breaths per
minute. A positive end expiratory pressure (PEEP) level of
5 cmH20 was achieved (or higher, as necessary) to reach an
oxygen saturation level equal to or higher than 95%.
The surgical technique employed is described in detail in
a previous publication (7). A brief summary follows.
Laparoscopy was performed using a peritoneal CO2 insufflation pressure between 12 and 15 cm H20. There was no
complication related to the trocar insertion. If a patient
presented with hypotension (defined as systolic arterial

RESULTS
The procedures were performed successfully. One patient
had an uncuffed tracheostomy tube and developed bilateral
capnothorax during surgery, which resulted in higher
ventilatory pressure. The condition was rapidly diagnosed
by evaluating the pulmonary pressure and was immediately
resolved after deflating the abdominal insufflation.
In another patient with a raised hemidiaphragm, a
Valsalva maneuver was performed to produce downward
force to help insert the permanent electrode. The Valsalva
maneuver produced hypotension that resolved after releasing the maneuver.
Another patient presented with capnothorax on a postoperative chest X-ray, and the decision was made to
perform pleural drainage with a pigtail catheter. After the
procedure, all of the patients recovered from anesthesia and
were returned to their previous respiratory support. The
patients were then transferred to the intensive care unit. On
the second postoperative day, the patients began conditioning training of the diaphragm muscle through the
intermittent use of the DPS for progressively longer time
periods (Figure 4).

Six months following the procedures, three patients
achieved continuous DPS use for 24 hours per day. A
patient who had relied on mechanical ventilation for 14
years achieved respiration for 6 hours each day with the
pacemaker. However, diaphragmatic stimulation was discontinued in this patient after the onset of uncontrolled
neuropathic pain. The fifth patient was unable to sustain
ventilation with the DPS.

recent study focused on the phrenic nerve motor point in
the diaphragm (12) and has created innovative applications
of diaphragm pacing in other populations, such as patients
with congenital central hypoventilation syndrome (13),
amyotrophic lateral sclerosis (14,15), and acute respiratory
failure (16).
Few studies are available on the perioperative management of patients with SCI. Although pre-medication can be
used, there is concern regarding the higher sensitivity to
medication in patients with SCI compared to the general
population, and a lower dose is thus recommended.
The specific challenges of this technique include performing the procedure without muscle relaxants, avoiding
hemodynamic instability secondary to pneumoperitoneum
and autonomic disorders that may be worsened by the
procedure (17). Neuromuscular blocking agents must be
avoided because they could interfere with the intraoperative
mapping of the diaphragm. Nevertheless, in our study, all
of the patients already had tracheostomies placed, and the
absence of neuromuscular blockers did not represent an
obstacle because tracheal intubation was unnecessary.
After venous induction, patients can present with severe
hypotension because of the absence of a sympathetic reflex
and relative hypovolemia. Before the induction of general
anesthesia, 500 to 1000 mL of crystalloid solution is
recommended. Patients with SCI also present a higher risk
of hypothermia; therefore, body temperature must be
monitored.
The phenomenon of autonomic dysreflexia is rare but is a
severe and life-threatening complication that can occur
during invasive surgical procedures in patients with spinal
cord injury. Autonomic dysreflexia is characterized by
disordered autonomic responses to certain stimuli below
the level of the lesion (18). Adequate fluid management,
careful perioperative management, and the proper depth of
anesthesia can successfully control autonomic dysreflexia.
We used BIS to monitor our patients to ensure the ideal
anesthesia depth and minimize the risk of autonomic
dysreflexia.
Although rare, the artificial pneumoperitoneum created
using CO2 can cause pneumothorax/capnothorax, a known
complication of laparoscopic surgery. CO2, a highly

DISCUSSION
Our study illustrates the perioperative management of
patients with severe SCI and chronic respiratory failure
who underwent insertion of DPS. This technique allows
patients to wean from mechanical ventilation.
Mechanical ventilation-related adverse events and safety
issues have been studied extensively in experimental (9)
and clinical trials (10,11) that emphasize the importance of
early extubation. The laparoscopic approach to DPS
implantation in our SCI patients was a safe and efficient
procedure with no severe complications.
Electric stimulation of the phrenic nerve for diaphragm
pacing in patients with SCI has been well documented
beginning with the pioneering work of Glenn (3). A more

Figure 3 - The clinical station and the diaphragmatic pacing
device.

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Diaphragmatic Pacing in Spinal Cord Injury
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CLINICS 2012;67(11):1265-1269

diffusible gas, can infiltrate into the pleural space through
congenital defects of the diaphragm or through parietal
pleura injuries that are caused by surgical manipulations. The
anesthesiologist must consider the possibility of capnothorax
when the patient presents with a sudden reduction in lung
compliance, an elevation in peak respiratory pressure and
end-tidal carbon dioxide (ETCO2). Hypotension and hypoxemia are uncommon events in this scenario. The resolution of
the capnothorax can be accelerated by using PEEP and
hyperventilation (19).
The most common complication that occurred in our
patients was capnothorax, which was observed in two cases.
These events were most likely related to the electrode
implantation procedure. Note that the incidence of capnothorax observed during the implantation of the NeuRx
DPS in patients with amyotrophic lateral sclerosis (ALS)
was lower than for the SCI patients. In the ALS cases,
capnothorax occurred in 16 of 86 (19%) patients and in the
SCI cases, capnothorax occurred in 21 of 50 (42%) patients
(20).
In another case series of six patients, Onders et al. (21)
reported the possible complications of wound infection and
intermittent aspiration. In our study, we observed incidence
of infection.
The efficacy of DPS has been suggested by Alshekhlee
et al., (22) who described a series of 26 patients who
received successful DPS implantations. In that study, 96%
of the patients were able to use the DPS, and 54% achieved
full-time pacing a median of 142 days following the
implantation.
The use of a diaphragm pacing system has changed the
medical outcome of SCI and chronic respiratory failure
patients who have long-term dependence on mechanical
ventilation (23). The improvement in tidal volumes and vital
capacity translates into a lower dependence on mechanical
ventilation and an increased quality of life. In addition, this
implantation technique is related to lower morbidity
compared to the traditional phrenic nerve pacing. This
improvement is due to the laparoscopic placement of the
DPS electrodes, which avoids bilateral thoracic access and
the potential risk of phrenic nerve damage (24).
Although the SCI patients presenting with chronic
respiratory failure and dependence on mechanical ventilation represent a high-risk group for perioperative complications, the successful implantation of a DPS was achieved in
all of the patients in our study.
Our results reinforce the premise that the laparoscopic
implantation of a DPS can be safely accomplished if certain
principles are followed: a) before surgery, the patients must
be evaluated regarding whether the diaphragm is stimulable and to determine that the phrenic nerves are intact; b)
optimal anesthetic management includes adequate premedication and avoiding over-sedation by adequate BIS
monitoring to allow early recovery and weaning from
ventilation; c) avoidance of neuromuscular blockers; d)
infection surveillance; and e) adequate post-operative ICU
care.
In conclusion, the implantation of a DPS is a safe and
efficient procedure with the potential to improve the quality
of life of patients who are dependent on mechanical
ventilation as a result of SCI. Adequate perioperative care
is essential to ensure the best results. Further experimental
trials are needed to assess the impact of the DPS technique
in these patients.

ACKNOWLEDGMENTS
This work was supported by the Fundac¸a˜o de Amparo a` Pesquisa do
Estado de Sa˜o Paulo (FAPESP – 2010/50785-6). A donation was also
received from Synapse Biomedical International.

AUTHOR CONTRIBUTIONS
Tedde ML conceived the study, collected the data, participated in the
analysis of the samples and drafted the manuscript. Vasconcelos-Filho P,
Hajjar LA, Flora GF, Okumura EM and Galas FR collected the data,
participated in the analysis of the samples and drafted the manuscript.
Almeida JP, Osawa EA and Fukushima JT participated in the analysis of
the samples and drafted the manuscript. Teixeira MJ, Jatene FD and Auler
Jr JO drafted and approved the manuscript’s final version.

cases and corticosteroid resistance in 4.8 to 27% of cases (46). This relapsing characteristic has led some authors to seek
diagnostic markers that can be used to differentiate
relapsing from non-relapsing patients, and some studies
have indicated that adults with early presentation of the
disease and high serum levels of IgE are more likely to
relapse (5,6).
A study in adult Chinese patients also found elevated
serum IgE levels in relapsing patients (7), and these findings
were explained by the fact that serum IgE levels reflected
immune dysfunction as well as B- and T-cell activation (7).
The absence of studies on this pathology in the Brazilian
population led us to conduct a retrospective study aimed at
identifying the clinical and laboratory characteristics of
primary onset MCD in a patient population over the age of
13 and establishing the clinical and laboratory characteristics of relapsing versus non-relapsing patients.

INTRODUCTION
Minimal change disease (MCD) is the leading cause of
nephrotic syndrome in children under the age of 10,
accounting for 90% of all cases. In adults, however, MCD
accounts for only 9-15% of primary glomerulopathy cases
(1-3), and studies in adults are scarce, likely due to the lower
disease incidence in this age group. Waldman et al.
observed that at the time of diagnosis, in addition to the
classic nephrotic presentation of glomerulopathy, 43% of
patients presented with hypertension, 17.8% had acute
kidney injury, and 29% had microscopic hematuria (4). It
was also observed that the treatment response was favorable. However, relapses are frequent and may range from
62.3 to 73.1%, with corticosteroid dependence in 12% of

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.

MATERIALS AND METHODS
We retrospectively evaluated patients who had been
diagnosed with nephrotic syndrome and MCD, as confirmed by renal biopsy, from 1979 to 2009 in two university

No potential conflict of interest was reported.

1271

Adolescent and Adult Minimal Change Disease
Dias CB et al.

CLINICS 2012;67(11):1271-1274

centers in the State of Sa˜o Paulo, Brazil (Hospital das
Clı´nicas da Faculdade de Medicina da Universidade de
Sa˜o Paulo and Universidade Estadual Paulista-School of
Medicine-Hospital das Clı´nicas of Botucatu).

RESULTS
We studied 63 patients with a median age at diagnosis of
34 (23-49) years, including 23 males and 40 females. The
biochemical data at diagnosis showed levels of serum
creatinine of 0.8 (0.7-1.3) mg/dL, serum albumin of 1.8
(1.5-2.3) g/dL, proteinuria of 7.2 (5.3-10.3) g/day, total
cholesterol of 423.0 (335.0-524.0) mg/dL and triglycerides of
214.5 (162.5-310.5) mg/dL. At the time of diagnosis, eight
(12.7%) patients presented with microscopic hematuria, 17
(27%) had hypertension and 17 (27%) had acute kidney
injury. The mean follow-up time for these patients was 24
(17-60) months.
The decision to treat and the choice of the treatment
regimen were made by the assistant nephrologist in
accordance with clinical criteria. In the initial treatment,
most patients (52, 82.5%) were treated only with prednisone at 1 mg/kg/day for eight weeks with gradual
tapering thereafter; three (4.7%) patients received cyclosporine; two (3.1%) were given prednisone and cyclophosphamide; and six (9.5%) received no immunosuppression
because they presented with spontaneous remission. After
the initial treatment, 55 (87.3%) patients demonstrated
complete remission, six (9.5%) presented with partial
remission and two (3.1%) were nonresponders. In the
patients with remission (n = 61), relapse occurred in 34
(54%) patients with a median of two (1-3) relapses per
patient during the follow-up period. Two of these patients
(3.1%) were considered dependent on immunosuppressive
medication.
Comparing the clinical presentation at diagnosis between
the groups of patients with relapse (n = 34) and without
relapse (n = 27), there was no difference in age (32.5 (22.546.0) vs. 34.0 (23.0-61.0) years, respectively), gender, clinical
presentation (hematuria, hypertension and acute kidney
injury) and follow-up time (Table 1). In the initial laboratory
data, there was a statistically significant difference only in
proteinuria, which was greater in the relapsing group than
in the non-relapsing group (8.8 (7.1-12.0) vs. 6.0 (3.6-7.3) g/
day, p = 0.001, respectively) (Table 1).
In a logistic regression, only proteinuria affected the
relapse frequency, with an odds ratio of 1.23 (p = 0.01; 95%
CI 1.04 to 1.46). However, the patient age at diagnosis and
the serum albumin level did not show any effect on relapse
(Table 2). For the patients with proteinuria .7 g/day (at
diagnosis), 27 (44.2%) were in the relapse group, and 11
(18%) were in non-relapse group (p = 0.002). In contrast, only
seven (11.4%) of the patients with proteinuria #7 g/day (at
diagnosis) were in the relapse group.
Sixty-five percent of patients received non-immunosuppressive agents for renin-angiotensin system-blocking therapy, and 45% received statin therapy. There was no
difference in the use of these medications between the
relapsing and non-relapsing patients.
At the end of the follow-up period, there were no
differences between the groups with and without relapse
regarding the levels of serum creatinine (0.8 (0.7-0.9) vs. 0.8
(0.6-0.9) mg/dL, respectively), proteinuria (0.1 (0.07-0.3) vs.
0.1 (0.06-0.3) g/day, respectively), triglycerides (97 (66-129)
vs. 102 (70-141.5)) mg/dL, respectively) and total cholesterol
(193.0 (158.0-216.0) vs. 191.0 (168.5-223.0) mg/dL, respectively). During this period, one patient in the relapse group
presented with loss of renal function but did not require
dialysis.

Inclusion Criteria
The following inclusion criteria were established: an
initial clinical presentation of nephrotic syndrome; a
diagnosis of MCD that was confirmed by renal biopsy
using conventional criteria with normal light microscopy
and negative immunofluorescence in a representative
sample; age greater than 13 years; and follow-up time of
at least 1 year.

Exclusion Criteria
Patients diagnosed with any systemic disease, such as
systemic lupus erythematosus or other autoimmune diseases, diabetes, or previous hypertension were excluded, as
were those with less than 1 year of follow-up. To confirm
these data, all of the patients were tested for ANA, viral
serology, and blood glucose, and they also received an
ophthalmological review or an echocardiogram.
From 1979 to 2009, 102 renal biopsies confirmed the
diagnoses of MCD in the proposed patient group. Thirtynine patients were excluded from the analysis; ten were
discharged to their original medical units and 29 missed
their follow-up appointments during this period.
The patient data were obtained through an analysis of
medical records and included clinical characteristics,
laboratory parameters, response to medical treatment and
relapses.
After an initial treatment, proteinuria ,0.3 g/day was
defined as complete remission; the reduction of baseline
proteinuria by .50% with a final value ,3 g/day was
defined as partial remission; and resistance was defined as
cases where no remission occurred after the use of
immunosuppressive medication for up to 16 weeks.
Relapse was defined as proteinuria values that returned to
.3 g/day after a remission period; immunosuppressive
medication dependence was defined as relapses occurring
within four weeks after withdrawal; and acute kidney injury
(AKI) was defined as an increase of 50% or more in the
baseline creatinine level.
Hematuria was defined as more than eight red blood cells
per high-power field. Hypertension was defined as systolic
blood pressure or diastolic blood pressure .139 mmHg or
90 mmHg, respectively, in two sequential readings.
This study was approved by the Nephrology Department
of the Universities of Sa˜o Paulo and Botucatu as well as the
university ethics committee.

Statistical Analysis
The continuous variable data were expressed as the
median with quartile intervals and percentages for categorical variables. Differences between the two groups were
evaluated using the unpaired Student’s t-test; when the
sample was not normally distributed, we applied the MannWhitney U-test. Categorical variables between groups were
evaluated using the chi-squared test. Logistic regression
was tested for a dependent variable (relapse) in relation to
the independent variables (patient age at diagnosis, serum
albumin and proteinuria). The level of statistical significance
was set at p,0.05.

remissions (9.5%). However, relapse is an important
characteristic of this disease, as it occurred in 54% of the
cases in our study. Nine patients (14.2%) experienced more
than two relapses during the follow-up period, and two of
these patients were considered dependent on immunosuppressive medication (Figure 1). Other studies have also
shown a high frequency of relapse, with values between
67.1 and 73.1% (4,5,11).
It would be beneficial to establish clinical and laboratory
characteristics to differentiate relapsing from non-relapsing
patients, with the aim of using steroids for a shorter period
of time and calcineurin inhibitors in the long term. In our
study, the patients who presented with proteinuria .7 g/
day at the time of diagnosis were more likely to experience
relapse. The clinical parameter most related to relapse in
other studies was age, indicating that younger patients
relapse more frequently than older patients (6,12); however,
this finding was not made in our sample population.
Glucocorticosteroids remain the first-line treatment for
relapse; however, in frequent relapsing and corticosteroiddependent patients, it is necessary to use other immunosuppressants to minimize the side effects of the prolonged use of
glucocorticosteroids. Eguchi A et al. demonstrated that the
combined use of low-dose cyclosporine and prednisone was
as effective at inducing remission as the use of prednisone
(1 mg/kg/day) alone (13). Even without proper knowledge
of the pathophysiology of MCD, treatment with glucocorticosteroids or calcineurin inhibitors can be effective at
inducing remission, but these drugs do not prevent relapses
in most cases. One of the theories regarding the pathophysiology of this disease is the dysfunction of podocytes with an
overexpression of angiopoetin-like-4 (ANGPTL4) and CD80
(14), the latter of which contributes to T-cell dysfunction (15).
In an experimental model, it was found that interleukin-13
acts as a potent stimulator of CD80 expression (16), and this
interleukin is also known for its role in allergic processes (17).
In summary, minimal change disease in adults, in
addition to its nephrotic characteristics, may present with
hematuria, hypertension, and AKI. The treatment response
and disease evolution are generally favorable, but there is a
major risk of relapse. According to the current study,
patients with proteinuria .7 g/day were at risk for relapse.
Although these findings are clinically relevant, interpretations should be made with caution because this was a
retrospective study and more trials are needed to support
the results.

The following complications occurred during the followup period. In the group without relapse, one patient
developed diabetes mellitus, and another developed melanoma. In the group with relapse, three patients developed
diabetes mellitus, two had osteoporosis, two had depression, and one developed obesity.

DISCUSSION
This study assessed retrospective data from two university centers over a 30-year period and found that
minimal change disease in adults affected a patient
population in their third decade of life (34 (23-49) years);
only nine patients were aged 60 years or older (data not
shown), and there was a slight predominance of females
(1.7/1). The disease evolution was favorable; however, one
patient developed chronic kidney disease, and one patient
aged 70 years was diagnosed with melanoma close to
glomerulopathy symptoms.
Our patients were similar to those observed in other
studies in terms of age at the onset of disease, gender and
the presence of hematuria at diagnosis (4,8). However, as
compared to other study protocols, our study evaluated
fewer patients with hypertension and proteinuria at
diagnosis and a higher frequency of patients with acute
kidney injury at diagnosis (4,8).
The clinical presentation of acute kidney injury in MCD is
associated with acute tubular necrosis in most cases and
occurs more frequently in older hypertensive patients (4,9).
In the present study, the AKI patients were older than those
without AKI (48.0 (34.0-60.0) vs. 29.0 (22.0-41.0) years,
p = 0.015), but there was no difference in hypertension,
proteinuria and serum albumin level between these groups
(data not shown).
The response to initial treatment with glucocorticosteroids for this pathology is generally high, approximately
80% (4,10), and our data revealed a total 96.8% remission
rate, including complete remissions (87.3%) and partial

AUTHOR CONTRIBUTIONS
Dias CB and Pinheiro CC assisted the patients and were responsible for the
statistical analysis. Silva VS and Hagemann R assisted the patients.
Woronik V and Barros RT contributed to the discussion and text revision.

OBJECTIVE: The aim of this study was to assess the IgE serum levels in juvenile systemic lupus erythematosus patients
and to evaluate possible associations with clinical and laboratory features, disease activity and tissue damage.
METHODS: The IgE serum concentrations in 69 consecutive juvenile systemic lupus erythematosus patients were
determined by nephelometry. IgG, IgM and IgA concentrations were measured by immunoturbidimetry. All patients
were negative for intestinal parasites. Statistical analysis methods included the Mann-Whitney, chi-square and
Fisher’s exact tests, as well as the Spearman rank correlation coefficient.
RESULTS: Increased IgE concentrations above 100 IU/mL were observed in 31/69 (45%) juvenile systemic lupus
erythematosus patients. The mean IgE concentration was 442.0¡163.4 IU/ml (range 3.5-9936.0 IU/ml). Fifteen of the
69 patients had atopic disease, nine patients had severe sepsis and 56 patients presented with nephritis. The mean
IgE level in 54 juvenile systemic lupus erythematosus patients without atopic manifestations was 271.6¡699.5 IU/ml,
and only nine of the 31 (29%) patients with high IgE levels had atopic disease. The IgE levels did not statistically
differ with respect to the presence of atopic disease, severe sepsis, nephritis, disease activity, or tissue damage.
Interestingly, IgE concentrations were inversely correlated with C4 levels (r = -0.25, p = 0.03) and with the SLICC/ACRDI score (r = -0.34, p = 0.005). The IgE concentration was also found to be directly correlated with IgA levels (r = 0.52,
p = 0.03).
CONCLUSIONS: The present study demonstrated for the first time that juvenile systemic lupus erythematosus
patients have increased IgE serum levels. This increase in IgE levels was not related to allergic or parasitic diseases.
Our results are in line with the hypothesis that high IgE levels can be considered a marker of immune
dysregulation.
KEYWORDS: Juvenile Systemic Lupus Erythematosus; IgE; Nephritis; Intestinal Parasite; Allergic Disease.
Liphaus BL, Jesus AA, Silva CA, Coutinho A, Carneiro-Sampaio M. Increased IgE serum levels are unrelated to allergic and parasitic diseases in patients
with juvenile systemic lupus erythematosus. Clinics. 2012;67(11):1275-1280.
Received For Publication on June 11, 2012; First review completed on July 20, 2012; Accepted for publication on July 20, 2012
E-mail: bernadete.liphaus@icr.usp.br
Tel.: 55 11 3898-1078

Of note, the polyclonal activation of B lymphocytes in SLE
patients results in the production of autoantibodies, particularly of the IgG and IgM classes, and, rarely, antinuclear IgE
autoantibodies and IgE immune complexes (1-4,7,9,10).
Indeed, IgE plays a central role in host immunity against
parasitic infections and in the pathogenesis of atopic diseases
(5,11). Recently, IgE has also been considered a biomarker for
immune dysregulation, as observed in patients with partial T
cell immunodeficiencies (5). To date, few studies have
evaluated the IgE levels in SLE patients (4,6,7). High total
IgE serum levels have been associated with disease activity
and nephritis in adult SLE patients (4,12-15). However, to our
knowledge, no studies have evaluated the association of IgE
with juvenile SLE (JSLE).
Therefore, the aim of this study was to assess total IgE
serum concentrations in JSLE patients and to evaluate
possible associations between increased IgE levels and
clinical and laboratory lupus features, disease activity and
tissue damage.

INTRODUCTION
Systemic lupus erythematosus (SLE) is a complex autoimmune disease with respect to its underlying genetics, and
it is characterized by the disruption of immune tolerance,
leading to a hyperactive Th2 response, polyclonal activation
of B lymphocytes, immune-complex deposition, and tissue
damage (1-5). In SLE patients, the Th2 response is evidenced
by the significant production of interleukins (ILs) 4, 5, and
10, which is similar to the interleukin profile in patients with
allergic disorders (4-8).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

1275

IgE in Juvenile SLE
Liphaus BL et al.

CLINICS 2012;67(11):1275-1280

modified techniques published by Rugai and Lutz,
Hoffman, Pons and Janer.

METHODS
Sixty-nine consecutive patients diagnosed with JSLE
based on the American College of Rheumatology (ACR)
revised classification criteria were enrolled in this study
(16,17). At the onset of disease, all patients were younger
than 16 years of age. This study was approved by the local
ethics committee, and informed consent was obtained from
all patients/parents participating in the study. The exclusion criteria included bacterial, viral, fungal or parasitic
infection at the time of study entry.

Statistical analysis
Continuous variables were analyzed using a MannWhitney test, and categorical variables were evaluated
using a chi-square or Fisher’s exact test, as appropriate
(27). Correlation analyses were performed using the
Spearman rank correlation coefficient (27). p-values ,0.05
were considered statistically significant.

RESULTS

Clinical evaluation and treatment

The mean age of the patients upon enrollment was
15.8¡3.7 years, and 58 of the 69 patients were female. The
mean disease duration and age at disease onset were
6.9¡3.6 and 8.8¡3.3 years, respectively. The clinical and
laboratory characteristics of the JSLE patients are presented
in Table 1.
Allergic manifestations were reported by 15 (21.7%) JSLE
patients; specifically, three had atopic dermatitis, nine
presented with allergic rhinitis and/or asthma and three
presented with both respiratory and cutaneous allergic
features. Severe sepsis was observed in nine (13%) patients.
Fifty-six (81.2%) patients presented with nephritis, 39 of
whom underwent renal biopsy. Of the patients who underwent renal biopsy, seven (17.9%) had focal proliferative
nephritis (class III), ten (25.6%) had diffuse proliferative
nephritis (class IV), and 17 (43.6%) had membranous
nephritis (class V).
The total IgE concentrations in the JSLE patients ranged
from 3.5 to 9936.0 IU/ml. Increased IgE concentrations
($100 IU/ml) were observed in 31 of the 69 (45%) patients,
and the mean IgE level was 442.0¡163.4 IU/ml (Figure 1).
The mean IgG, IgM and IgA concentrations were
1387.9¡557.4, 115.8¡59.0 and 207.7¡152.3 mg/dl, respectively.
The IgE levels ranged from 6.1 to 9936.0 IU/ml in the
JSLE patients with atopic disease, 3.5 to 9936.0 IU/ml in
patients with active disease (SLEDAI 2K$4), 3.5 to
2934.0 IU/ml in patients with severe sepsis and 3.5 to
4920.0 IU/ml in patients with nephritis. In JSLE patients
without atopic manifestations (n = 54), the mean IgE level

Patients and parents were systematically inquired regarding the following allergic manifestations: atopic dermatitis,
acute or chronic urticaria, allergic rhinitis, asthma, and drug
and food reactions. The presence of infection (recurrent
pyogenic infections, mycobacteriosis, fungal infections,
herpes zoster and/or severe sepsis) was also assessed.
Severe sepsis was defined according to the international
pediatric sepsis consensus conference definitions (18).
Medical records were evaluated for patients clinical
findings, which included cutaneous, hematological, renal,
musculoskeletal and neuropsychiatric manifestations. The
renal histologic class according to the World Health
Organization classification criteria was also registered (19).
Disease activity and disease-related tissue damage were
determined for each patient upon enrollment based on the
SLE Disease Activity Index 2K (SLEDAI 2K) and the
Systemic Lupus International Collaborating Clinics/ACR
Damage Index (SLICC/ACR-DI) scores, respectively (2022). Disease activity was arbitrarily defined as equivalent to
a SLEDAI 2K score$4.
All patients were prescribed glucocorticoid therapy, and
fifty-eight patients were taking one of the following
immunosuppressive agents: intravenous cyclophosphamide, azathioprine, methotrexate, or mycophenolate mofetil.

Laboratory evaluation
Total serum IgE concentrations were determined by
nephelometry (Dade Behring/Siemens, Deerfield, USA).
According to recommendations by the manufacturer and
several previous studies on IgE levels, the cut-off value for
an elevated IgE level was set at 100 IU/ml (23-26). Serum
IgG, IgM and IgA levels were determined by immunoturbidimetry (Roche Diagnostics, Indianapolis, USA).
The following laboratory parameters were also analyzed:
complete blood cell count, urinalysis, and erythrocyte
sedimentation rate, determined using the Westergren
method; C reactive protein, determined by nephelometry;
and serum complement components C3 and C4, determined
by nephelometry. Serum levels of C1q were determined by
radial immunodiffusion. Antinuclear antibodies (ANAs)
were detected by indirect immunofluorescence in HEp-2
cells, and anti-dsDNA antibodies were determined by both
indirect immunofluorescence on Crithidia luciliae and by
quantitative ELISA.
Three consecutive stool samples were collected from each
patient. Stool analyses were performed by a blinded,
qualified technician by microscopic examination for the
detection of protozoan oocysts, cysts, helminthic eggs, and
larvae using techniques published by Faust et al., as well as

DISCUSSION
The present study showed that JSLE patients have an
increased serum IgE concentration regardless of the
presence of atopic manifestations or parasitic disease.
SLE pathogenesis is complex, and there remain controversies concerning the involvement of immunoglobulin E in
the pathogenesis of the disease (1-5). The dysregulation of
immune tolerance results in aberrant Th2 responses and
polyclonal activation of B lymphocytes along with the
production of autoantibodies, including those of IgE isotype
(1-5,10). The human Th2 immune response, characterized by
the significant production of IL-4, IL-5, IL-10, and IgE, is
mainly observed in atopic diseases and in some parasitic
infections; however, elevated IgE production has also been
observed in patients with partial T cell immunodeficiencies
and autoimmune diseases (5,7,8).
Although IgE synthesis is tightly controlled by regulatory T
cells, B cells, and cytokines, the role of immunoglobulin E in
autoimmune diseases has not been fully elucidated (11). The
most striking observation reported in the literature is that
elevated IgE production and allergic and autoimmune
manifestations frequently occur in patients with partial T cell
immunodeficiencies even though elevated IgE levels and
autoimmune and inflammatory diseases are traditionally
associated with hyperactivity of the adaptive immune system
(5). Moreover, mouse models have recently highlighted that
an increased IgE level frequently accompanies different
partial T cell immunodeficiencies that result in autoimmunity
(5). Taken together, these observations suggest that IgE levels
increase when there is an imbalance between the immunogenic and tolerogenic signals in effector T cells; thus, elevated
IgE levels can be considered a biomarker of immune
dysregulation (5).
Although some authors have postulated that IgE is not
related to connective tissue disease pathogenesis, others
have claimed that IgE plays an essential role in connective
tissue disorders (7,28). The latter authors state that, through
the release of vasoactive mediators from basophils and mast

cells, IgE can cause increased vasopermeability, which may
be important in causing the deposition of circulating
immune complexes in glomerulonephritis pathogenesis
(28,29). The demonstration of increased IgE serum levels
in SLE patients with renal involvement and the detection
of IgE immune complex deposition in renal biopsies
further implicate IgE in the pathogenesis of lupus
nephritis (13,29,30). High IgE levels have also been
reported in adult SLE patients without nephritis, suggesting that IgE may have a role in SLE disease and not only
in nephritis (4,12,14,15,31,32). Furthermore, antinuclear
and anti-DNA autoantibodies of the IgE isotype were
observed in adult SLE patients, and these IgE autoantibodies did not correlate with the serum IgE concentration
(4,9,10,33). Anti-IgE IgG autoantibodies have also been
observed in SLE patients with lymphoadenopathy, articular involvement and anti-DNA antibodies (9). In addition,
increased IgE serum levels were observed in children of
mothers with lupus, regardless of the presence of allergic
disease in the mothers (34). Another study showed
elevated IgE levels in male SLE patients compared with
female patients (35). To the best of our knowledge, this
was the first study to demonstrate increased IgE levels in
JSLE patients.
SLE and allergic disorders share certain immunological
abnormalities (6,7) because the prevalence of IgE-mediated
and/or IgE-associated disorders, such as allergic reactions
to drugs, atopic dermatitis, asthma, allergic rhinitis and
allergic conjunctivitis, has been reported to be elevated in
patients with lupus (6,12,36,37). In contrast, recent studies
observed a similar prevalence of IgE-related disorders in
SLE subjects compared with patients without SLE (30,38-41).
In our study, 15 (21.7%) JSLE patients had at least one
respiratory and/or cutaneous allergic manifestation, which
is similar to the rate of atopic disorders found in healthy
subjects (40,42).
The current study showed that JSLE patients harbor
higher IgE concentrations in a manner independent of the
presence of allergic disease. Our observations are in line
with recent reports stating that patients with lupus are not
at an increased risk of IgE-mediated allergic disorders.
However, our observations differ from those of previous
studies reporting evidence of an increased incidence of
atopic conditions in SLE patients (12,13,38,41).
We also observed increased IgE serum levels in JSLE
patients independent of the presence of parasitic infections.

However, the IgE serum concentrations varied widely,
which may be due to differences in environment- and
patient-specific factors, such as contact with antigens, race,
gender and age. Witting et al. (24) showed that an IgE level
of 100 IU/ml is the upper-limit cutoff for elevated IgE
diagnostic sensitivity and specificity for all patient cohorts.
Thus, one possible limitation of this study is the lack of
comparison with age- and gender-matched healthy controls.
Regarding lupus activity, patients with severe active
disease (SLEDAI 2K $10) had IgE levels at least two times
higher than the IgE levels of patients with inactive disease,
although this difference was not statistically significant.
Interestingly, IgE concentrations correlated inversely with
C4 levels, which could suggest that the complement
cascade was activated and its components were consumed,
as shown by the reduction of C4 levels, which is also
considered a marker of disease activity. The latter statement that IgE levels could be a marker of disease activity
requires further investigation because the correlation
between IgE and C4 levels was relatively weak. Although
serum IgE levels have been reported to vary according to
disease activity in adult SLE patients (14,15,31,39), our
findings could not confirm this correlation in a definitive
manner in patients with JSLE.
Interestingly, IgE serum concentrations were inversely
correlated with SLICC/ACR-DI scores, suggesting a protective role for increased IgE levels. This finding is contrary
to current knowledge that patients with active disease have
high IgE levels and are consequently at a higher risk of
organ damage. In addition to the observed direct correlation
between IgE and IgA serum levels, the inverse relationship
between IgE levels and SLICC/ACR-DI scores supports the
hypothesis that increased IgE levels can be considered a
marker of immune dysregulation, which may be important
in the generation of immune complexes.
Lupus nephritis, which has various histological patterns
and variable clinical outcomes, is one of the most important
SLE-associated morbidities. Although the pathogenic
mechanism in each histological type of nephritis remains
unclear, some findings point to a role for both Th1 and Th2
immune responses in renal damage (7,28). Th1 cytokines
have been related to diffuse lupus nephritis, while Th2
cytokines have been associated with membranous lupus
nephritis (7,13,28-30). In the present study, 81.2% of the JSLE
patients had lupus nephritis, and IgE levels $100 UI/ml
were observed in 36.2% of these patients. However, the

mean IgE levels were similar in patients with and without
nephritis, as well as in patients with severe renal disease. This
observation is in contrast with the published works with adult
SLE patients, which showed a significant correlation between
serum IgE concentration and nephritis activity, and previous
reports, which showed that IgE renal deposits in lupus patients
correlate with a poor prognosis (13,30,39).
All patients with JSLE were taking glucocorticoids, and
the down-regulation of allergic inflammation could have
been associated with the use of this medication, as
glucocorticoids can increase IL-10 gene transcription and
decrease both IL-4 and IL-5 gene transcription (43).
Finally, despite the variety of factors that can influence
IgE production, the present study demonstrated for the first
time that JSLE patients have higher IgE concentrations,
suggesting that increased IgE levels could play a role in
lupus pathogenesis. However, the specific mechanisms
underlying the elevation of IgE levels in children with
lupus remain to be clarified, and further studies are needed.

ACKNOWLEDGMENTS
This work was supported by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do
Estado de Sa˜o Paulo) under grant number 08/58238.

AUTHOR CONTRIBUTIONS
All authors drafted the manuscript, critically reviewed the manuscript for
intellectual content, and approved the final version to be published.
Liphaus BL contributed to the study conception and design, data analysis
and interpretation. Jesus AA contributed to the data acquisition, analysis
and interpretation. Silva CA contributed to data interpretation and
critically reviewed the manuscript. Coutinho A contributed to the study
conception and data interpretation. Carneiro-Sampaio M contributed to
data interpretation and manuscript intellectual content.

Post-traumatic ventricular septal defect (VSD) is an
uncommon, if not rare, sequel of penetrating or blunt
trauma, post-surgical contusion, and myocardial infarction
(1). Open-heart surgery has been the standard method of
treatment (2), but percutaneous closure has emerged as a
new technique to address this important and challenging
surgical problem. In this procedure, an Amplatzer VSD
Occluder has usually been employed (3), or occasionally,
an Atrial Septal Defect (ASD) Occluder has been used (4).
We present our experience with percutaneous closure of
post-traumatic VSDs utilizing a PDA Occluder (Lifetech
Scientific (Shenzhen) Co., LTD, Guangdong, China). The
PDA Occluder is shown in Figure 1A.

Three previously healthy patients who were stabbed in the
left anterior chest presented with knives lodged in the fourth
left intercostal space that moved synchronously with the
heartbeat. The patients were awake but restless and in severe
pain, with cardiac tamponade, dyspnea, hypotension, and
elevated central venous pressure. The results of patients’
physical examinations are described in Table 1. The knives
were surgically removed, and perforations of the right
ventricle were repaired with pledgeted-reinforced mattress
sutures. On the second post-operative day, a loud holosystolic
murmur was detected in all three of the patients. Transthoracic
echocardiography revealed a large VSD in the muscular
septum of each patient, and their pulmonary/systemic flow
ratios (Qp/Qs) were over 1.7. Pulmonary arterial pressure was
mildly elevated. The transthoracic echocardiography data are
listed in Table 2 and one patient’s echocardiography image is
shown in Figure 1B.

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.

Percutaneous closure
Cardiac catheterization with a femoral approach was
performed using local anesthesia. A pigtail catheter was

introduced into the left ventricle. A left ventricular
angiogram disclosed a significant left-to-right shunt across
a large defect in the muscular ventricular septum. The
pigtail was withdrawn, and a multi-purpose catheter was
guided from the left ventricle into the right ventricle and
advanced through the VSD into the pulmonary artery. A
second catheter was introduced into the femoral vein and
advanced into the pulmonary artery. The defect in the first
patient was closed with a Muscular VSD Occluder. The
device became trapped during placement of the occluder
because the right plate had an inappropriate configuration.
The device was retrieved.
A PDA Occluder was selected to close the defect. The
deviceâ&#x20AC;&#x2122;s size was similar to that of the VSD or ASD
Occluder, judged by the maximum diameter viewed by
echocardiography and angiography. The delivery sheath
was introduced into the left ventricle and ascending aorta in
a loop. The PDA Occluder was then mounted and inserted
into the delivery sheath (Figure 1C). After release of the
device, left ventriculography revealed no left-to-right shunt.
In the following two patients, the PDA Occluder was our
primary choice.

RESULTS
The transthoracic echocardiogram revealed that the
device was in place with no left-to-right shunt. Ventricular
size and function were normal. Pulmonary arterial pressure
was normal (Figure 1D). In addition, 320-slice computerized
tomography showed that the Occluder was well placed and
had normal morphology (Figure 1E).

DISCUSSION
Penetrating cardiac injuries are among the most common
causes of violent death (5). Cardiac injury occurs in
approximately 20-30% of cases of major chest trauma (6).
In the majority of cases, the injuries are fatal. Approximately
20% of patients are alive when they arrive in the hospital,
and up to 70% of these patients survive to hospital
discharge (7). Survival depends upon rapid diagnosis and
immediate treatment.
A VSD due to penetrating cardiac injury can occur
directly, due to perforation of the septum, or indirectly,
following injury of an epicardial coronary artery with

necrosis and subsequent rupture. VSD is the most common
complication of penetrating heart injuries. There are reports
of delayed VSD presentation in which initial echocardiography was normal, but subsequent echocardiograms revealed a
defect (8). The most common location of a post-traumatic
VSD is at the apex (9). Surgical repair of a traumatic VSD can
incur significant morbidity and mortality (10).
Surgical repair may not be feasible immediately after
injury because of the patient’s poor clinical condition. In
addition, an increase in the defect’s size can result from
progressive tissue necrosis due to injury of the septal
coronary arteries after perforation of the interventricular
septum. Delay allows time for the development of fibrotic
scarring on the rims of the defect, facilitating delineation on
transthoracic echocardiography, which is important for
optimal device selection and fixation (2).
Percutaneous closure has been successful in children with
congenital VSD, approximately 90% of which are in the
perimembranous septum (11). In most studies, the
Amplatzer Muscular VSD Occluder and ASD Occluder
were employed. Complete closure using the Amplatzer
Muscular VSD Occluder or ASD Occluder is achieved in
92% of patients with a perimembranous VSD and in 95% of
patients with a muscular VSD (11).
In a previous report, an Amplatzer device was successfully implanted in 16 of 18 patients with a post-infarction
VSD; however, the 30-day-mortality was as high as 28%
(12). In a group of mixed cases, successful VSD closure was
reported in 30 of 32 patients (13). Prior our experience, only
sporadic cases of successful percutaneous closure of traumatic VSD had been reported (3,9). For repair of posttraumatic VSDs, an Amplatzer Muscular VSD Occluder (3)
or an ASD Occluder is usually chosen (4). The use of a PDA
Occluder in this type of procedure has not been previously
reported. We successfully repaired three post-traumatic
VSDs with percutaneous with PDA occluders. A Muscular
VSD Occluder initially employed in the first patient failed
for two reasons. The post-infarction VSD was nearly
angulated (Figure 1B), but the congenital VSD or ASD is
straight. The Amplatzer Muscular VSD Occluder or ASD
Occluder is designed for congenital heart disease, so the
length was insufficient (the perforations in our three
patients were more than 9 mm long, while the metal waist
length of the Amplatzer Muscular VSD Occluder implant is
only 4 mm). The Amplatzer Muscular VSD Occluder and
ASD Occluder, which have two discs, are difficult to release
in an angulated pathway. However, the PDA Occluder has
only one disc. The higher blood pressure in the left ventricle
serves the PDA Occluder’s left disc; thus, it is safe. During
the follow-up periods of the three patients (the longest for
five years), the PDA Occluders remained securely
implanted in each case.
Use of the PDA Occluder in a VSD closure has several
advantages in comparison to the Amplatzer Muscular VSD
Occluder or the ASD Occluder. The PDA Occluder cannot
cause ventricular outflow tract obstruction because it only
has a left disc. In addition, the metal waist of a PDA
Occluder is both more flexible and softer than other
Occluder implants, so it is more easily released and molded.
The implant moves synchronously with the heartbeat.
Fatigue damage of the metal material (14) occurs under
the action of the dynamic load, and the implant’s main

cause of failure is fatigue rupture. The fatigue life of the
PDA Occluder is increased in comparison to the other
implant types.
Our experiences indicate that percutaneous closure of a
post-traumatic VSD using a PDA Occluder device is
feasible, safe and effective. However, more experience is
warranted before recommending widespread use of the
PDA Occluder in this procedure.

ACKNOWLEDGMENTS
The authors wish to thank Dr. Joseph K. Perloff, Professor of Medicine and
Pediatrics at UCLA Medical Center, for his careful revision of the
manuscript.

AUTHOR CONTRIBUTIONS
Zhu J, Zhang Y and Xia F contributed to the written paper. Xi EP, Zhu
SB, Yin GL, Liu Y and Dong YQ contributed to patient treatment. Zhu SB conceived the study. Xi EP is the first surgical doctor for
these patients. Zhu J was responsible for the manuscript first draft.

OBJECTIVES: The phosphatidylinositol 3-kinase/AKT axis is an important cell-signaling pathway that mediates cell
proliferation and survival, two biological processes that regulate malignant cell growth. The phosphatidylinositol 3kinase CA gene encodes the p110a subunit of the phosphatidylinositol 3-kinase protein. There are phosphatidylinositol 3-kinase CA mutations in several types of human tumors, and they are frequently observed in breast cancer.
However, these mutations have not been investigated in Brazilian breast cancer patients.
METHODS: PCR-SSCP and direct DNA sequencing were performed to identify phosphatidylinositol 3-kinaseCA exon
9 and exon 20 mutations in 86 patients with sporadic breast cancer. The relationships between PIK3CA mutations
and patient clinicopathological characteristics and survival were analyzed. The presence of the TP53 mutation was
also examined.
RESULTS: Twenty-three (27%) of the 86 primary breast tumors contained PIK3CA mutations. In exons 9 and 20, we
identified the hotspot mutations E542K, E545K, and H1047R, and we identified two new missense mutations
(I1022V and L1028S) and one nonsense (R992X) mutation. Phosphatidylinositol 3-kinase CA exon 20 mutations were
associated with poor overall survival and TP53 gene mutations.
CONCLUSIONS: Phosphatidylinositol 3-kinase CA mutations are common in tumors in Brazilian breast cancer
patients, and phosphatidylinositol 3-kinase CA and TP53 mutations are not mutually exclusive. Phosphatidylinositol
3-kinase CA exon 20 mutations are associated with poor survival, and they may be useful biomarkers for identifying
breast cancer patients with aggressive tumors and for predicting the response to treatment with PI3K pathway
inhibitors.
KEYWORDS: Breast Neoplasm; PIK3CA; TP53; Mutation; Prognosis.
Mangone FR, Bobrovnitchaia IG, Salaorni S, Manuli E, Nagai MA. PIK3CA exon 20 mutations are associated with poor prognosis in breast cancer
patients. Clinics. 2012;67(11):1285-1290.
Received for publication on July 9, 2012; First review completed on July 17, 2012; Accepted for publication on July 23, 2012
E-mail: nagai@usp.br
Tel.: 55 11 3893-3013

tumors, including glioblastomas, gastric cancers, lung
cancers, ovarian cancers, hepatocellular carcinomas, endometrial carcinomas, brain cancers, and breast cancers (3).
The majority of PIK3CA mutations cluster in hotspot regions
in exon 9 (the helical domain) and exon 20 (the kinase
domain). The most common missense mutations change
amino acid residues E542 and E545 to lysine in the helical
domain and change H1047 to arginine in the kinase domain.
Functional studies suggest that these particular PIK3CA
mutations lead to increased PI3K activity (6,7).
The frequency of PIK3CA mutations in breast cancer
ranges from 16.4 to 45% (3,8-10). However, the association
between PIK3CA mutations and specific clinicopathological features of breast cancer is still a matter of debate.
Furthermore, the relationship between the presence of
PIK3CA mutations in breast cancer patients and overall
survival (OS) and disease-free survival (DFS) is controversial. Some studies have found that breast cancer patients
with PIK3CA gene mutations have improved OS and DFS
rates compared with breast cancer patients lacking such
mutations (9,11-13). Conversely, other studies have found

INTRODUCTION
The phosphatidylinositol 3-kinase (PI3K)/AKT signaling
pathway plays an important role in cellular processes, such
as proliferation, differentiation, survival, and migration
(1,2). Alterations in the components of this signaling
pathway, including gain-of-function mutations in the p110
catalytic subunit of PI3K, have been identified in a wide
spectrum of human cancers (3,4). Class I PI3Ks are
heterodimers composed of catalytic (p110) and regulatory
(p85) subunits involved in regulating cell division and in
tumorigenesis (5,6).
The PIK3CA gene comprises 20 exons encoding the p110a
catalytic subunit. This gene is mutated in a wide range of

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

1285

PIK3CA mutations in breast cancer patients
Mangone FR et al.

CLINICS 2012;67(11):1285-1290

that the presence of PIK3CA mutations is correlated with
poor outcome (14-16).
In the present study, we identified mutations in exons 9
and 20 of the PIK3CA gene in primary breast tumors from
Brazilian breast cancer patients, and we analyzed the
relationship between mutational status and patient clinicopathological features and outcomes.

Mutation analysis by polymerase chain reactionsingle stranded conformation polymorphism (PCRSSCP) and direct DNA sequencing
Two sets of primers were used to amplify exon 9 (forward:
59-CCAGAGGGGAAAAATATGACA-39; reverse: 59-CATTTTAGCACTTACCTGTGAC-39) and exon 20 (forward: 59CATTTGCTCCAAACTGACCA-39; reverse: 59-TGAGCTTTCATTTTCTCAGTTATCTTTTC-39) of the PIK3CA gene.
The PCR products were separated using the GeneGelTM
Excel 12.5/24 Kit (GE Healthcare, Sweden) according to
the manufacturer’s instructions. Gels were stained using the
DNA Silver Staining Kit (GE Healthcare) according to the
manufacturer’s instructions. Samples exhibiting differences
in gel band mobility were cloned (TOPO-TA CloningH Kit,
Invitrogen) and then sequenced using a MegaBACE 1000
automatic sequencer (Amersham Biosciences) and the ET
Dye Terminator Kit (Amersham Biosciences). All sequences
were analyzed using Mutation Surveyor software v3.2
(SoftGenetics).

MATERIALS AND METHODS
Tumor samples and genomic DNA extraction
Samples from 86 primary breast tumors were obtained
from breast cancer patients diagnosed at the Hospital do
Cancer A. C. Camargo, Sa˜o Paulo, Brazil, from February 1993
to March 1998. The median follow-up time was 63.3 months
(range, 25 to 78 months). None of the patients had received
any medical treatment related to their breast cancer before
the biopsy/mastectomy procedure. After surgical excision,
biopsy specimens were immediately frozen and stored in
liquid nitrogen until DNA extraction. Histopathological
review of the tumor slides was performed to confirm the
diagnosis. All tumors were classified according to the World
Health Organization Histological Typing of Breast Tumors
classification, and the clinical stage of each patient was
determined according to the 5th Edition of the UICC TNM
classification of malignant tumors. The tumors were all
infiltrating ductal carcinomas. The median age of the patients
at the time of diagnosis was 55 years (range, 26 to 85 years).
The patient and tumor characteristics are shown in Table 1.
Tissue specimens were ground to a powder under liquid
nitrogen using a Frozen Tissue Pulverizer (Termovac
Industries, Copiague, N.Y.), and high-molecular-weight
DNA was extracted as previously described (17). This study
was approved by the Hospital das Clı´nicas da Faculdade de
Medicina da Universidade de Sa˜o Paulo Ethics Committee.
All subjects were given information about the study and
provided written informed consent.

Statistical analysis
Fisher’s exact test and Spearman’s rho correlation were
used to assess the association and the direction of the
association, respectively, among categorical variables. OS
and DFS rates were calculated based on the Kaplan-Meier
method, and the curves were compared using the log-rank
test. OS and DFS rates were determined from the day of the
diagnosis to the date of death or to the date on which
recurrence was detected, respectively.
Statistical analyses were performed using IBM SPSS
Statistics 19.0, 2010 (SPSS Chicago, IL). Differences were
considered significant when the p-value was less than 0.05.

RESULTS
We investigated mutations in exon 9 and exon 20 of the
PIK3CA gene in 86 primary breast tumors by performing
SSCP analysis and DNA sequencing. Of the 86 tumors, 23
(27%) exhibited PIK3CA mutations: 13% in exon 9 and 14%
in exon 20. Table 2 lists the PIK3CA variants identified by
DNA sequencing. We characterized seven non-synonymous
variants, two of which were new (I1022V, L1028S); three
synonymous variants, two of which were new (S541S,
L1028L); one new stop codon-gain variant (R992X); and
one previously known stop codon-loss (X1069W) variant.
Figure 1 shows representative electropherograms of the
PIK3CA variants characterized in the primary breast tumors.

Figure 1 - Representative eletropherograms of the PIK3CA mutations characterized in the breast cancer biopsy samples in this study. (A)
Known mutations and (B) new mutations.

(PIK20mut) and TP53 mutations (Fisherâ&#x20AC;&#x2122;s test p-value = 0.05,
Spearmanâ&#x20AC;&#x2122;s correlation = 0.03, r = 0.253; Table 3).
We also tested whether PIK3CA mutations were associated with patient OS or DFS. A comparison of patients
who had tumors with or without PIK3CA mutations
revealed no significant differences in cancer-specific survival. On the other hand, when patients were grouped
according to the presence of PIK3CA helical domain (exon
9) or kinase domain (exon 20) mutations, the presence of
exon 20 mutations was associated with poorer OS (p = 0.026)
and DFS (p = 0.079) (Table 4 and Figure 2). We further
analyzed the relationship between survival and exon 20
mutations by conducting Kaplan-Meier analyses. We found
that patients with tumors harboring exon 20 mutations had
a significantly shorter mean OS and DFS compared with
patients lacking exon 20 mutations (median OS: 24.1 months
and not reached, respectively, p = 0.007; median DFS: 15.9
months and not reached, respectively, p = 0.025) (Table 4
and Figure 2).

New variants were considered mutations, as they were not
present in the paired normal tissue of the same patients
(data not shown). The frequency of the hotspot mutation
E545K was 8.1%, corresponding to 63.6% of the helical (exon
9) mutations. The other common helical (exon 9) mutation,
E542K, was observed in only one case. The kinase (exon 20)
hotspot mutation H1047R was observed at a frequency of
14%, representing 91.7% of the PIK3CA exon 20 mutations.
Next, we investigated whether PIK3CA mutations were
associated with breast cancer development and progression.
The demographic and clinicopathological characteristics of
patients with tumors containing PIK3CA mutations were
compared with those of patients with tumors lacking
PIK3CA mutations. There were no statistically significant
differences between the clinicopathological features or
steroid hormone receptor status in patients with or without
PIK3CA mutations (Table 3).
Using a data set of TP53 mutations published previously
by our group (17), we evaluated whether any of the 73
patients had both PIK3CA and TP53 mutations. None of
the tumors with exon 9 PIK3CA mutations (PIK9mut) also
contained TP53 mutations. In contrast, we observed a
correlation between the presence of exon 20 PIK3CA mutations

DISCUSSION
No previous study has investigated the frequency and
spectrum of PIK3CA mutations in primary tumors from

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Table 3 - Association between the presence of PIK3CA mutations and patient and tumor characteristics.
Variable

significant associations between PIK3CA mutations and
steroid hormone (estrogen and/or progesterone) receptor
status in breast cancer patients (13,14,18,19), while others
failed to find such associations (12,15). Although the
association between PIK3CA mutations and steroid hormone receptor status did not reach statistical significance,
we observed a higher frequency of PIK3CA mutations in
estrogen receptor-positive tumors compared with receptorâ&#x20AC;&#x201C;
negative tumors, mainly in exon 9.
The association between PIK3CA mutations and breast
cancer patient survival remains controversial. In the present
work, we found that kinase domain (exon 20) mutations
were strongly associated with poorer OS and DFS. Various
studies have reported that the presence of PIK3CA mutations is associated with good prognosis (11,13), is associated
with poor prognosis (14,16), or has no survival effect (18,20)
in breast cancer patients. Kalinsky et al. (13) found a direct
association between the presence of mutations in the C2,
helical, or kinase functional domains and better DFS or OS.
They also found that the H1047R mutation was strongly
associated with the absence of lymph node metastasis (13).
Similarly, Maruyama et al. (11) described a positive

Brazilian breast cancer patients. In this study, we identified
PIK3CA mutations in primary breast tumors from a group of
Brazilian breast cancer patients and correlated these mutations with patient clinicopathological features and outcomes. The observed frequency of PIK3CA mutations was
27%, which is in accordance with similar studies that have
examined the frequency of exon 9 and 20 mutations
(frequency range, 16.4 to 45%) (3,8-10). This result indicates
that PIK3CA mutations are quite common genetic events in
tumors in Brazilian breast cancer patients. The frequency of
the most common missense activating mutations (E542K,
E545K, and H1047R) in the primary breast tumors was
82.6%, the same rate previously reported in the literature
(11). We also identified three new PIK3CA variants, two
missense variants, and one nonsense variant. These variants
were considered mutations, as they were not present in the
paired normal tissue of the same patients (data not shown).
In our analysis of the relationship between PIK3CA
mutations and patient clinicopathological characteristics,
we found no significant correlations between PIK3CA
mutations and patient age, clinical stage, tumor size, or
lymph node metastasis. Some previous studies showed

correlation between the presence of mutations in any
domain of the PIK3CA gene and better relapse-free survival.
Taken together, these studies suggest a protective role for
these mutations. On the other hand, similar to our study,
two other studies reported that exon 20 mutations were
associated with poorer OS (14,16). It is difficult to compare

these studies because of the studies’ population heterogeneity and because there may have been differences in the
therapeutic strategies not mentioned in the publications.
Mutations in TP53 and PIK3CA are frequent in breast
cancer (21). In the present study, we found a positive
correlation between the presence of PIK3CA exon 20 and

TP53 mutations, with four samples exhibiting mutations in
both genes. This result suggests that the presence of these
mutations is not mutually exclusive, as was proposed by
Boyault et al. (19). We previously reported that patients with
tumors harboring TP53 mutations affecting amino acids
involved directly in DNA or zinc binding had a poor
prognosis (17). Interestingly, in this study, we found that the
presence of PIK3CA exon 20 mutations could be used to
stratify patients into distinct prognostic groups, regardless
of whether a TP53 mutation was present.
In summary, this is the first study to report that PIK3CA
mutations are common in tumors in Brazilian breast cancer
patients. We found that PIK3CA exon 20 mutations were
significantly associated with TP53 mutations, indicating that
PIK3CA mutations and TP53 mutations are not mutually
exclusive. Our finding that PIK3CA exon 20 mutations were
associated with more aggressive breast cancer and poor
outcomes, regardless of the treatment regimen, has important clinical implications.

ACKNOWLEDGMENTS
This study was funded by a grant from the Departamento de Cieˆncia e
Tecnologia-Ministe´rio da Sau´de/Conselho Nacional de Desenvolvimento
Cientı´fico e Tecnolo´gico (grant number 577587/2008-0 DECIT/CNPq)
and by a CNPq grant (grant number 305408/2009-7).

OBJECTIVES: The objectives of this study were to verify the degree of anxiety, respiratory distress, and health-related
quality of life in a group of asthmatic patients who have experienced previous panic attacks. Additionally, we
evaluated if a respiratory physiotherapy program (breathing retraining) improved both asthma and panic disorder
symptoms, resulting in an improvement in the health-related quality of life of asthmatics.
METHODS: Asthmatic individuals were assigned to a chest physiotherapy group that included a breathing retraining
program held once a week for three months or a paired control group that included a Subtle Touch program. All
patients were assessed using the Diagnostic and Statistical Manual of Mental Disorders IV, the Sheehan Anxiety
Scale, the Quality of Life Questionnaire, and spirometry parameter measurements.
RESULTS: Both groups had high marks for panic disorder and agoraphobia, which limited their quality of life. The
Breathing Retraining Group program improved the clinical control of asthma, reduced panic symptoms and
agoraphobia, decreased patient scores on the Sheehan Anxiety Scale, and improved their quality of life. Spirometry
parameters were unchanged.
CONCLUSION: Breathing retraining improves the clinical control of asthma and anxiety symptoms and the healthrelated quality of life in asthmatic patients.
KEYWORDS: Asthma; Physiotherapy; Panic; Breathing Retraining; Anxiety.
Laurino RA, Barnabe´ V, Saraiva-Romanholo BM, Stelmach R, Cukier A, Nunes MP. Respiratory rehabilitation: a physiotherapy approach to the control
of asthma symptoms and anxiety. Clinics. 2012;67(11):1291-1297.
Received for publication on June 12, 2012; First review completed on July 18, 2012; Accepted for publication on September 19, 2012
E-mail: ppatro@usp.br
Tel.: 55 11 3061-7317

There is increasing recognition that psychological factors
influence the onset and course of asthma. Furthermore, there
is a significant correlation between asthma and negative
emotions, particularly anxiety and depression (2).
Anxiety is a very common illness, with a prevalence
estimated at up to 20% of the adult population. Anxiety has
demonstrated effects on indicators of quality of life (5).
Panic disorder and agoraphobia are among the many
anxiety disorders. Agoraphobia is the intense fear of finding
oneself in crowded places where there may be a perception
of difficulty to escape.
Previous cross-sectional community-based studies have
provided evidence for a relatively specific association
between the prevalence of asthma and panic disorder
(2,6). Both anxiety and depression are known to influence
the quality of life in asthmatics, and both put stress on the
health care system (3). A previous longitudinal study
showed that asthma increases the risk of panic, anxiety,
and depression (6). More complex models have described
asthma as an organic disease that is highly vulnerable to
psychological influences (7,8). Mood disorders were identified in 53% of asthmatic patients and in 34.9% of nonasthmatics (9).

INTRODUCTION
The relationship between asthma and anxiety is wellestablished. Symptoms, such as respiratory discomfort, are
highly common in both panic disorder and in asthma. There
is evidence that breathing retraining helps to control the
symptoms of asthma and panic attacks with consequent
improvement in an asthmatic’s quality of life (1-3). However,
there is no strong evidence that a Chest Physiotherapy (CPT)
program, specifically the breathing retraining technique,
helps to reduce the symptoms of anxiety and improve
asthma control (1).
Asthma is a chronic inflammatory disorder of the airways
in which many different types of cellular elements play roles.
In susceptible individuals, this inflammation causes recurrent episodes of wheezing, breathlessness and coughing (4).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

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According to the 2009 Physiotherapy Guidelines (12),
breathing retraining (as part of CPT) incorporates a reduction
in the respiratory rate and/or tidal volume with relaxation
training, which helps to control the symptoms of asthma and
is recommended as level 1++ scientific evidence (Grades A
and B). Breathing retraining includes instruction in pursedlip breathing and coordinated breathing with respiratory
exercises and has the added benefit of reducing anxiety and
stress (13).
Physiotherapists have advocated chest physiotherapy for
the management of breathing disorders (13,14). In a cohort
of asthmatics, a randomized controlled trial of breathing
retraining and relaxation led to a significant reduction in
respiratory symptoms and improvement in quality of life
(15). In another randomized controlled trial, asthmatic
patients trained in diaphragmatic breathing had clinically
relevant improvements in their quality of life, even nine
months after the intervention (16). Other studies (17,18)
reported effective reductions in the number of symptoms, the
frequency of attacks and degrees of depression and anxiety,
and improvements in respiratory parameters.
This study aimed to assess the degree of anxiety and
respiratory distress and the quality of life in a group of
asthmatic patients who have previously experienced panic
attacks. We also demonstrated that a respiratory physiotherapy program (breathing retraining) may improve both asthma
and panic disorder symptoms, resulting in an improvement
in the health-related quality of life of asthmatics.

Table 1 - Preliminary patient demographic and
spirometry data for both the Breathing Retraining Group
(BRG) and the Subtle Touches group (STG). Eighty percent
of the BRG patients and 72.2% of the STG patients had
moderate/severe asthma. The BRG patients had a higher
frequency of previous smoking. Both groups had high
scores for panic disorder and agoraphobia, as indicated
by the appropriate scales. The BRG and the STG had the
same Daily Symptom Diary median scores.
Subject characteristics

MATERIALS AND METHODS
The Sheehan Disability Scale (19) is a three-item selfreported scale that measures the severity of the disability in
the areas of work and family life, home responsibilities, and
social activities or hobbies. Each of these three areas is
scored on a Likert scale of ten points (a score of 0 is ‘‘not at
all impaired,’’ 5 is ‘‘moderately impaired’’ and 10 is ‘‘very
severely impaired’’). The scale provides a measurement of
total functional disability (range 0-30) and has been shown
to have appropriate internal reliability and validity (20). It
has previously been used in studies of panic disorder (21).
The Quality of Life questionnaire assesses an individual’s
well-being, complements traditional health and clinical measures, and captures the wider impact that asthma has on
physical, psychological, and social life. The specific instrument
we used for the determination of quality of life has been
validated for use in clinical trials (22). It assesses four domains:
activity limitation, symptoms, emotional function, and environmental stimuli (22). The Quality of Life questionnaire assesses
physical limitation, the severity and frequency of symptoms,
adherence to treatment, and psychological factors.

Study design and eligibility
In this prospective study, we used specific and validated
methods for the quantification of anxiety symptoms in two
randomly selected samples of asthmatic patients from the
Asthma Clinic of the Pneumology Department of the
Hospital das Clı´nicas da Universidade de Sa˜o Paulo.
The study was approved by the Ethics Committee of the
institution, and informed written consent was obtained from
each patient. Asthmatic patients who had well-controlled
symptoms and received regular inhaled corticosteroids and
long-acting bronchodilators for at least one month were
included in the study according to the criteria of the Global
Initiative for Asthma (2). The inclusion criteria for the study
were: 1. at least three symptoms of panic and agoraphobia; 2.
persistent fear of public places or open areas or the need to be
removed from fear situations that trigger the crisis; and 3.
fulfillment of the criteria of asthma according to American
Thoracic Society (ATS).
All patients underwent a clinical exam and were evaluated
with the DSM-IV-R to establish panic and agoraphobia
symptoms, the Sheehan Anxiety Scale, the Quality of Life
questionnaire, forced vital capacity (FVC), forced expiratory
volume in one second (FEV1), forced expiratory flow 25-75%
(FEF 25-75%), and FEV1/FVC ratio (FEV1/FVC). Patients were
evaluated at the beginning of the study (Table 1 and Table 2) and
twelve weeks later [twice during the study (T = 0 and T = 12)].
Patients recorded daily symptoms/signs and rescue
salbutamol use in their diaries. The peak expiratory flow
rate (PEFR) was monitored with a portable device (Mini –
Wright, Clement Clark International, Harlow, Essex,
England) once a week for three months. Patient diaries
detailing symptoms and rescue salbutamol use were
collected at three months.

Experimental groups
Thirty-eight asthmatic patients with a history of panic
symptoms entered this case-controlled study and were
randomly assigned (1:1 for two groups) to a breathing
retraining group (BRG, n = 20) or a control group that received
Subtle Touch (STG) (n = 18). The Subtle Touch technique
controls for the presence and the action of the physiotherapist
as potential confounding factors (Figure 1).

Exercise protocol
The same physiotherapist performed all of the physical
therapy maneuvers. The subjects were seen individually, once a

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Table 2 - The evolution of parameters expressed as median values (25%-75%). At the start of the study, both groups
presented with equally compromised health-related quality of life scores and the same levels of panic and asthma
symptoms. Final measurements indicated improvements in all domains.
Variations in the domains of quality of life and symptoms in asthmatic patients.
Group

VDAT - Variation of the domain: adherence to treatment between groups; VDSF - Variation of the domain: severity/frequency between groups; VDPL Variation of the domain: physical limitation; VDP - Variation of the domain: psychosocial; VDSE - Variation of the domain: social-economic; VRS - range of
symptoms; PD - panic disorder. * Comparing the beginning of the experiments to the end of the protocol.

week, in an outpatient setting in which each patient underwent
a total of 30 minutes of Subtle Touch or breathing retraining.
Breathing retraining consists of six repetitions of each the
following physiotherapeutic exercises: 1. pursed-lip breathing
associated with a lying relaxed posture; 2. manual expiratory
passive therapy maneuvers; 3. diaphragmatic breathing; 4.
hiccup inspiratory maneuvers; 5. postural orientation; and 6.
twenty repetitions of Pompage (maneuvers performed for the
muscle fascia).
Subtle Touch, also called Calatonia, is a Jungian method
(23) that was developed by Petho Sandor, who demonstrated that continued treatment with Subtle Touch
promotes the reduction of anxiety and depression and
leads to a stable mood. In this study, we used only the
physical aspects of Subtle Touch. This technique uses both
hands simultaneously on both sides of the thorax. The
Subtle Touch technique is performed in three steps: 1. a
gentle pressure is imposed on the skin with hands or
fingertips for three breaths, with a slow decompression
after the last one; 2. soft touches are performed during the
next three breaths; and 3. hands or fingertips maintain only
slight contact with the skin and remain still for three more
respiratory cycles.
Due to the limited sample size, a confidence interval of
95% and a maximum estimate error of 20% were used. A
control group (STG) was used to reduce sample limitations.

RESULTS
Table 1 summarizes the clinical and demographic data of
38 patients who completed the study. Forty-five patients
were initially recruited. Seven patients did not complete the
study; one died before randomization. Of the six remaining
patients who did not complete the study, four were in the
BRG. Three of these four patients did not attend all the
physiotherapy sessions, and one suffered an asthma
exacerbation. Two patients in the STG did not complete
the study. One of these two patients suffered an asthma
exacerbation, and the second patient did not attend all the
scheduled sessions (Figure 1).
The spirometry data from the beginning of the trial
revealed that, in both groups, the patients had values
indicative of moderate or severe asthma (Table 1). There
were no significant differences in spirometry measurements
(FVC, FEV1, FEF25-75%, FEV1/FVC) between the two
groups throughout the study. Interestingly, the peak flow
rate was significantly different between both groups.
We observed a progressive and significant increase in the
peak flow rate in the BRG (p#0.05); it remained stable with a
slight downward trend in the STG (Figure 3A). The use of a
b2 agonist (salbutamol) as a relief medication (Figure 3B)
decreased over time after the sixth week of the study, which
was significant for the BRG (p#0.05).
Patients from both groups had high levels of agoraphobia
and panic disorder at the start of the study. Statistically
significant reductions in panic disorder symptoms (according to the DSM-IV-R) (p#0.05) and agoraphobia (p#0.05)
were observed only in the BRG (Figure 2A). There was no
difference in the same parameters when multiple analyses
were performed comparing the BRG with the STG. Panic
scores, according to the Sheehan scale, exhibited significant
decreases for both the BRG and the STG (p#0.05)
(Figure 2B).
Initially, the asthmatic patients who participated in this
study had substantial limitations in their quality of life, despite
receiving notably good clinical treatment and being considered stable. The variability in the Quality of Life questionnaire
occurred throughout the four domains (Table 2). Over the
course of multiple analyses, there were statistically significant
decreases in the values for the ‘‘physical limitation’’ domain
(p#0.05) and the ‘‘gravity/frequency’’ domain (p#0.05). There
was also an improvement in the psycho-social factors domain

Statistical analysis
Statistical analysis was performed using Sigma Stat
software (Jandel Corp, San Rafael, CA). The data are
presented as means and standard deviations (SD) for age,
FEV1, and the Daily Symptom Scale. The scores for panic
disorder (PD), agoraphobia (AG), and Sheehan’s Disability
Scale are expressed as medians and ranges. The distribution
of asthma severity and the frequency of smoking are
expressed as percentages. All data were analyzed intragroup and inter-group and were compared to the values at
the beginning and end of the study. A paired Student’s t-test
was used to compare the initial and final intra-group values.
One-way analysis of variance was used to compare the
experimental groups before and after experimental exercise.
Values of beta-2 agonist use, spirometry parameters and
peak flow were compared using a two-way analysis of
variance (ANOVA).

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Figure 1 - Study design.

for the two groups, although this improvement was not
statistically significant (p,0.01). At the end of the protocol, only
the BRG showed significant differences in the ‘‘adherence to
treatment’’ (p#0.05) and ‘‘social economic’’ (p#0.05) domains.

increased and consumption of salbutamol decreased. The Subtle
Touch technique also led to improvements in these parameters,
but not to the same extent as breathing retraining.
Subjectively, the authors noted in their clinical experience
that episodes of breathlessness caused quality of life changes
in asthmatics, including a high attention deficit and a sense of
hopelessness regarding their disease prognosis. Additionally,
patients could learn about conscious control over breathing to
overcome psychological aspects. A review of the literature and
some existing publications on chest physiotherapy, anxiety,
and asthma revealed that there was a theoretical basis for a
more organized treatment approach.

DISCUSSION
In this study, the three-month chest physiotherapy program
improved the clinical control of asthma and the quality of life and
decreased the symptoms of panic and agoraphobia in a group of
asthmatics with high anxiety scores at baseline. Spirometry
parameters remained unchanged, the daily peak flow values

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Figure 2 - At the end of the study, the scores for agoraphobia and panic disorder symptoms (Figure 2A) were significantly reduced only
in the BRG group (p,0.05). According to the Sheehan scale, panic scores (Figure 2B), were significantly decreased for both the BRG and
the STG (p,0.05). There were no differences in the same parameters when multiple analyses were performed comparing the BRG with
the STG.

(28). Panic and fear are established risk factors for worse
asthma-related morbidity, independent of objective measures of pulmonary function (29). Generalized panic or fear
may affect the self-management of asthma by influencing
decisions about the use of rescue medication and the
avoidance of activities. The general tendency to respond
with anxiety to perceived threats in the environment (trait
anxiety) might lead patients to treat their emotional distress
with short-acting b2-agonists, particularly if they confuse
respiratory symptoms of anxiety for asthma (30). However,
excessive use of short-acting b2-agonists might also contribute to greater levels of anxiety because of the side-effects
of these medications, such as tachycardia and tremor
(31,32). The association between generalized panic or fear
and avoidance of activities due to asthma may reflect

Patients enrolled in this study had symptoms of asthma
for more than twenty years; 76.3% had histories of
hospitalization during these years, and 34.2% had been in
an intensive care unit. All of the patients had high scores for
panic and agoraphobia. As was observed by other authors,
these data suggest that asthma severity can be associated
with anxiety and vice versa (24).
Some authors have sought to understand and clarify the
relationships between the respiratory system, mental state,
and personality (25,26). There are many reports and studies
linking anxiety disorders and depression to respiratory
dysfunction (5,26,27) and respiratory disorders in anxious
patients without lung disease (2,16).
A high level of psychiatric comorbidities was shown in
patients with severe asthma or difficult-to-control asthma

Figure 3 - Weekly peak flow rate measurements (3A) and daily use of b2 agonist (3B) were monitored over three months. Peak flow rate
progressively and significantly increased in the BRG (p,0.05) but remained stable in the STG (Figure 3A). In the BRG, the use of b2
agonist (salbutamol) as a relief medication (Figure 3B) decreased over time, particularly after the sixth week (p,0.05).

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and anxiety with physical therapy. The benefits observed in
the twelve weeks of this study required observation for a
longer period of time and were consistent.
In conclusion, breathing retraining improves the clinical
control of asthma symptoms, anxiety symptoms, and the
health-related quality of life in asthmatics. In particular,
substantial benefits were observed in severely compromised
patients.

agoraphobic anticipation and may extend well beyond
adaptive aversion to asthma triggers (33).
This behavior may manifest as a greater demand for
health care, higher numbers of missed work and school
days, and increased morbidity and mortality rates. This
behavior can also result in the unnecessary use of
medications.
There are few reports that use chest physiotherapy and
relaxation techniques to control respiratory disease and
anxiety in asthmatics. The relationship between respiratory
and psychological disorders remains unclear.
A systematic review concluded that chest physiotherapy
may have some potential benefits (34). Two separate
randomized controlled trials of patients with asthma
demonstrated that chest physiotherapy and relaxation
significantly improved health-related quality of life and
identified significant reductions in asthma symptoms
(15,35). These results also call for further studies to
demonstrate the efficacy of breathing retraining on asthma
(36). In accordance with the literature, these new concepts in
asthma management should help reduce hospitalizations
and emergency room visits, increase adherence to treatment
and improve patient quality of life (37).
This study focused on non-pharmacological interventions
in addition to the recommended drug treatment for
controlling asthma. A breathing retraining program led to
a statistically significant effect in controlling symptoms of
anxiety and airway obstruction, with improvements in peak
flow rate and the use of lower amounts of salbutamol
without significant changes in the FEV1 of both groups. The
peak flow rate depends on the patient’s effort and the
strength and speed of expiratory muscle contraction.
Breathing retraining promotes biomechanical reorganization and improves muscle function, which leads to a
significant improvement in peak flow independent of an
improvement in airway obstruction (22).
A control group (Subtle Touch) was introduced to reduce
possible confounding variables, such as the impact of a
therapist and the weekly contact with patients during visits.
Even in the Subtle Touch group, a generalized significant
improvement was observed. Subtle Touch treatment reduced
scores for panic disorder and agoraphobia, although to a
lesser degree than in the breathing retraining group.
Assuming that all patients were under optimal drug therapy,
the superior outcome observed for the BRG compared to the
STG is evidence for the benefits of breathing retraining to
control asthma and anxiety symptoms.
For the first time, we have demonstrated that the Subtle
Touch technique reduces the psychiatric symptoms associated
with asthma. These results are consistent with the hypothesis by
Sandor. Subtle Touch is a deep relaxation technique that leads
to the regulation of muscle tone and the promotion of physical
and psychological rebalancing of the patient. Essentially, Subtle
Touch therapy is performed by applying gentle stimuli in areas
of the body where there are particularly high concentrations of
nerve receptors. By promoting muscle relaxation, such techniques may lead to improvements in respiratory muscle function.
According to the Jungian proposal, the Subtle Touch technique
works by reducing anxiety.
This study noted an increase in peak flow values and a
decrease in consumption of salbutamol for patients who
underwent physiotherapy. These results are consistent with
those of other studies (17) that show an effective reduction of
symptoms, frequency of attacks, and degree of depression

ACKNOWLEDGMENTS
This work was supported by the following Brazilian scientific agencies:
FAPESP, CNPq, PRONEX-MCT, and FFM.

AUTHOR CONTRIBUTIONS
Laurino RA conceived the study, was the first to observe the anxious
attitudes of asthmatic patients under her physiotherapeutic care, designed
the study with the other investigators (Dr. Nunes - advisor, Dr. Cukier, Dr.
Stelmach), was responsible for the physical therapy applied to all subjects in
the Breathing Retraining Group (BRG) or the Subtle Touches Group
(STG), performed the data analyses and drafted the first version of the
manuscript. Laurino RA learned the Subtle Touch technique with a
specialist who supervised her during the study. Barnabe´ V provided
assistance during the study design and analysis of the data obtained
through the application of the Sheehan Anxiety Scale Questionnaires and
provided substantial assistance during the preparation and review of the
manuscript. Saraiva-Romanholo BM participated in the study design and
helped in the data analysis of spirometry parameters and peak flow rates,
provided substantial assistance during the statistical analysis and writing of
the manuscript. Stelmach R is one of the founders of the Group of
Assistance to the Asthma Patients of Hospital Clı´nicas da Faculdade de
Medicina da Universidade de Sa˜o Paulo, actively participated in the study
design, was responsible for the medical care of the patients throughout the
study and provided substantial contribution to the manuscript. Cukier A is
one of the founders and is currently the Chief of the Group of Assistance to
the Asthma Patients of Hospital Clı´nicas da Faculdade de Medicina da
Universidade de Sa˜o Paulo, actively participated in the study design, was
responsible for the medical care of the patients throughout the study and
provided substantial contribution to the manuscript. Nunes MP provided
assistance to the study design with the other investigators, contributed and
reviewed the statistical analysis with the help of the other researchers and
provided substantial contribution to the manuscript. All the authors have
read and approved the manuscript final version.

OBJECTIVE: The development of osteoporosis is associated with several risk factors, such as genetic structures that
affect bone turnover and bone mass. The impact of genetic structures on osteoporosis is not known. Plasminogen
activator inhibitor type-1 regulates the bone matrix and bone balance. This study assessed the correlation between
plasminogen activator inhibitor type-1 gene 4G/5G polymorphisms and osteoporosis in a population of Turkish
women.
METHODS: A total of 195 postmenopausal female patients who were diagnosed with osteoporosis (Group I) based
on bone mineral density measurements via dual-energy x-ray absorptiometry and 90 females with no osteoporosis
(Group II) were included in this study. Correlations between PAI-1 gene 4G/5G polymorphisms and osteoporosis
were investigated through the identification of PAI-1 gene 4G/5G polymorphism genotypes using the polymerase
chain reaction.
RESULTS: No significant differences in the genotype and allele frequency of 4G/5G plasminogen activator inhibitor
type-1 polymorphisms were observed between the two groups, and both groups exhibited the most frequently
observed 4G5G genotype.
CONCLUSION: No correlation between the development of osteoporosis in the female Turkish population and 4G/
5G plasminogen activator inhibitor type-1 gene polymorphisms was observed.
KEYWORDS: Osteoporosis; Polymorphism; Plasminogen Activator Inhibitor Type-1 (PAI-1) Gene; Bone Mineral
Density; Turkish Women.
Ozgen M, Turgut Cosan D, Doganer F, Soyocak A, Armagan O, Gunes HV, et al. Relationship between plasminogen activator inhibitor type-1 (PAI-1)
gene polymorphisms and osteoporosis in Turkish women. Clinics. 2012;67(11):1299-1302.
Received for publication on September 14, 2012; First review completed on September 16, 2012; Accepted for publication on September 20, 2012
E-mail: merihsarhus@hotmail.com.tr
Tel.: 0 90 532-5837099

INTRODUCTION
Osteoporosis is a systemic skeletal disease that is
characterized by an increase in bone fragility due to a
decrease in bone mass and the deterioration of bone
microarchitecture (1). This disease is especially prevalent
in the elderly population, and it is a significant public health
issue that reduces patient functioning and quality of life. An
improved understanding of the risk factors for osteoporosis
is important for the diagnosis, maintenance, and treatment
of this significant disease (2,3).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

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CLINICS 2012;67(11):1299-1302

obtained genomic DNA was maintained at 4 ˚C. The PAI-1
polymorphism gene region was amplified in a thermal
cycler (Sacem Life Technologies, Peltier-based Thermal
Cycler SCM 96G, Turkey) using 25 ml of a PCR mixture
containing 0.5 ml DNA, 10X PCR Buffer (Biolabs, New
England), 0.2 mmol/L dNTPs (Sigma, Germany), 1.25 U
Taq polymerase (Biolabs, New England), 50 pmol of 4G- or
5G-specific primer, 50 pmol of downstream primer, and
2.5 pmol of upstream primer. The following primers
(Metabion, Germany) were used: 5’-GTC TGG ACA CGT
GGG GG-3’ for the 5G allele, 5’-GTC TGG ACA CGT GGG
GA-3’ for the 4G allele, 5’-TGC AGC CAG CCA CGT GAT
TGT CTA G-3’ for the downstream primer, and 5’-AAG
CTT TTA CCA TGG TAA CCC CTG GT-3’ for the upstream
primer (positive control). The PCR mixture was subjected to
35 cycles for 60 sec at 94 ˚C, 30 sec at 54 ˚C, and 40 sec at 72 ˚C
following the initial denaturation for 3 min at 94 ˚C. These
PCR products were processed in 2% agarose gel and
analyzed under UV light (Labwork, Cambridge, United
Kingdom). The 4G and 5G alleles were defined according to
a 139-bp DNA fragment of the general downstream primer
that was produced during the PCR process. Samples that
produced a 139-bp band from the 4G primer and that did
not produce a 139-bp band from the 5G primer were
considered a homozygous 4G genotype. Samples that
produced a 139-bp band from the 5G primer but that did
not produce a 139-bp band from the 4G primer were
considered a homozygous 5G genotype. Samples that
produced a 139-bp band from both primers were considered
a heterozygous 4G5G genotype.

polymorphic loci in approximately 12.22 kb on chromosome
7q22. The 4G/5G insertion/deletion is the most investigated
polymorphism, which is 675 base pairs (bp) upstream of the
transcriptional start site. This polymorphism regulates the
expression of the PAI-1 gene (9,13,14).
The correlation of the PAI-1 4G/5G insertion/deletion
polymorphism with several diseases, such as coronary
artery disease, asthma, hypertension, stroke, obesity, rheumatoid arthritis, and osteoarthritis, has been investigated
previously (15-21). However, the contribution of PAI-1
insertion/deletion variations (4G/5G) to osteoporosis has
not been investigated in the Turkish population. This study
investigated the correlation between PAI-1 gene polymorphisms and osteoporosis in Turkish females.

MATERIALS AND METHODS
Subjects
Postmenopausal females who were admitted to the
Osteoporosis Clinic of the Physical Medicine and Rehabilitation Department of Eskisehir Osmangazi University
(Turkey) were informed of the study, and patients who opted
for inclusion in the study were evaluated. Patients who were
diagnosed with parathyroid, thyroid, liver, and rheumatological diseases that affect bone metabolism; patients with a
history of malignancy or surgically induced menopause; and
patients who used drugs affecting bone metabolism (e.g.,
corticosteroids, anticonvulsants, and heparin) during the
clinical and laboratory assessments were excluded from the
study. Erythrocyte sedimentation rate, complete blood count,
serum alkaline phosphatase, calcium, phosphorous, serum
glutamic oxaloacetic transaminase, serum glutamic pyruvic
transaminase, gamma-glutamyl transpeptidase, blood urea
nitrogen, creatinine, glucose, uric acid, albumin, total protein,
urine calcium/creatinine, thyroid-stimulating hormone, parathyroid hormone, cortisol and vitamin D levels were
measured prior to the study. A total of 285 patients satisfied
the study criteria and were included in the study. The age,
height, weight, and body mass index (BMI) of the participants
were evaluated. All participants underwent dual-energy xray absorptiometry (DEXA) evaluations, and 195 postmenopausal females were diagnosed with osteoporosis based on
this assessment (Group I). Ninety patients without osteoporosis were included in the control group (Group II). All
participants provided informed consent in compliance with
the study protocol (#2009/229), which was approved by the
Ethics Committee of the Medical Faculty of Eskisehir
Osmangazi University (Turkey).

Statistical analysis
The data were evaluated using SPSS Version 20 software
(IBM Corp. Armonk, New York, USA). The continuous
variables were not normally distributed based on the
Shapiro-Wilk test for normality. The Mann-Whitney U test
was implemented for the comparison of the two groups.
Medians (quartiles) are provided as descriptive statistics.
The Pearson chi-square test was conducted for categorical
variables. N and % values are provided. A p,0.05 was
considered statistically significant.

RESULTS
This study investigated the effect of the PAI-1 gene 4G/
5G polymorphisms on the development of osteoporosis in
Turkish women. The study groups are listed in Table 1. No
significant differences in the genotype and allele frequency
of the 4G/5G PAI-1 polymorphism were observed between
the groups (p = 0.619 and p = 0.361, respectively). However,
the most frequent genotype, 4G5G, was observed in both
groups. The 4G5G genotype was 39.49% in Group I and
42.22% in Group II. The 4G and 5G allele frequencies ranged
from 47.4 - 52.6% in Group I and 43.3 - 56.7% in Group II.

DISCUSSION
Osteoporosis is characterized by low bone mass, an
increase in bone fragility, deterioration in bone microarchitecture, and an increase in the risk of fracture (1). Some
metabolic changes, such as those that occur due to a lack of
estrogen, immobilization, metabolic acidosis, hyperparathyroidism, and systemic and local inflammatory diseases,
affect the osteoclast count and activity associated with bone

Sample collection and determination of PAI-1
genotypes
Genomic DNA isolation was performed using the saltextraction method in 10 ml of peripheral blood that was
collected in EDTA tubes for the analysis of 4G/5G
polymorphisms in the promoter region of PAI-1. The

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PAI-1 gene polymorphism and osteoporosis
Ozgen M et al.

Our results suggest that the 4G/5G PAI-1 polymorphism
cannot be used as a marker for the development of
osteoporosis in Turkish women. However, this result may
not be applicable to all populations when gene pools,
lifestyles, and gene-environment interactions in various
populations are considered. Therefore, multi-centered studies on different populations and in different gene regions
in larger samples are required to establish the correlation
between the 4G/5G PAI-1 polymorphism and osteoporosis.

turnover (22). Prostaglandins, insulin-like growth factors
(IGFs), interleukins (IL-1, IL-6, and IL-11), tumor necrosis
factor (TNF), and several local factors in bone, such as
transforming growth factor (TGF), also contribute to the
regulation of bone formation and resorption (22,23).
TGF-b is an anabolic factor that increases extracellular
matrix production and the expression of various types of
collagen and proteoglycans (24,25). TGFb1 polymorphisms
may be significantly relevant in BMD and the occurrence of
fracture (24,26). PAI-1 is known to have a regulatory effect
on matrix components, including TGF-b, matrix c-carboxyglutamic acid (Gla) protein, and osteocalcin. Therefore,
PAI-1 may affect bone matrix biology and significantly
regulate bone remodeling (10). PAI-1 levels are regulated by
a 4G/5G insertion/deletion polymorphism (13).
This study investigated the relationship of the 4G/5G
polymorphism, which regulates PAI-1 as an inhibitor of the
plasminogen activator system, with osteoporosis in Turkish
women.
A relationship between the PAI-1 4G/5G gene polymorphism and diseases, such as coronary artery disease,
hypertension, stroke, and obesity, has been reported
previously, but this polymorphism is not related to asthma,
rheumatoid arthritis, and osteoarthritis (15-21). Genetic
variations occur in populations. Previous studies have
investigated the PAI-1 4G/5G insertion/deletion polymorphism in the Turkish population, but its relationship
with osteoporosis has not been investigated; this relationship was examined in our study for the first time.
No differences in the PAI-1 4G/5G genotype were
observed between the postmenopausal osteoporotic patients
and the healthy control group. The role of common
variations of COLIA-1, TGFb-1, and PAI-1 genes in early
postmenopausal osteoporotic Caucasians and healthy
women was previously investigated by Hubacek et al.,
who observed no significant difference in the PAI-1 4G/5G
genotype between osteoporotic patients and the healthy
control group, which is consistent with our study. However,
the 4G4G genotype was more common in the osteoporotic
patient group compared with the control group (27).

OBJECTIVE: Postsurgical abdominal adhesions are common, serious postoperative complications. The present study
compared the usefulness of 4% icodextrin and canola oil in preventing postoperative peritoneal adhesions.
METHODS: Twenty-four Wistar albino rats were divided into three groups. Following a laparotomy, a serosal
abrasion was made by brushing the cecum, and 3 mL of 0.9% NaCl, 4% icodextrin, or 3 mL of canola oil were
intraperitoneally administered for the control, icodextrin, and canola oil groups, respectively. The abdomen was
then closed. All of the rats were sacrificed at day 10. Macroscopic, histopathological, and biochemical evaluations
were performed. The results were statistically analyzed using Kruskal–Wallis and ANOVA tests.
RESULTS: Macroscopic analyses revealed that both canola oil and 4% icodextrin reduced adhesion formation, but
the difference was not statistically significant (p = 0.17). The histopathological examinations revealed no significant
differences in terms of giant cell, lymphocyte/plasmocyte, neutrophil, ICAM1, or PECAM1 scores. However, both
canola oil and 4% icodextrin significantly reduced fibrosis (p = 0.025). In the canola oil group, the histiocytic
reactions were significantly increased (p = 0.001), and the hydroxyproline levels were significantly lower than those
in the other groups (p = 0.034).
CONCLUSIONS: In the present study, canola oil was determined to be superior to 4% icodextrin in lowering
hydroxyproline levels and increasing histiocytic reactions. Considering these results, we believe that canola oil is a
promising agent for preventing adhesion formation.
KEYWORDS: Peritoneum; Peritoneal Adhesion; Icodextrin; Canola Oil; Hydroxyproline; Experimental Study.
Yigitler C, Karakas DO, Kucukodacı Z, Cosar A, Gu¨lec B, Akin ML. Adhesion-preventing properties of 4% icodextrin and canola oil: a comparative
experimental study. Clinics. 2012;67(11):1303-1308.
Received for publication on May 29, 2012; First review completed on July 12, 2012; Accepted for publication on July 16, 2012
E-mail: cyigitler@hotmail.com / cyigitler@gata.edu.tr
Tel.: 90 532 6224824

large number of inflammatory and anti-inflammatory processes. Peritoneal adhesions are initiated by tissue damage
and can cause a coagulation cascade over the course of
several hours. Following coagulation, the inflammation
phase begins within the first few postoperative days. Cell
seeding, proliferation, migration and matrix deposition occur
in the first week postoperatively. Lastly, the matrix remodeling phase lasts over a period of weeks to months (5).
Several studies have aimed to reduce the frequency of this
commonly encountered condition. Progesterone, soybean
oil, aloe vera gel, vitamin E, methylene blue, and amniotic
membrane have been frequently used in experimental
studies of peritoneal adhesion prevention (5-12). These
materials are most commonly instilled and/or lavaged into
or around the peritoneal cavity in a liquid, gel or spray
form. Some of these substances have been used to inhibit
adhesion formation (i.e., coagulation, inflammation and
matrix formation), and others separate the peritoneal
surfaces.
A corn starch derivative, icodextrin is a water-soluble
branched glucose polymer; its monomers are linked by

INTRODUCTION
Postoperative peritoneal adhesions are major complications in abdominopelvic surgery, occurring in 60-93% of
patients (1,2). These adhesions can result in major postoperative complications, such as intestinal obstruction,
infertility, and chronic pelvic pain and can require readmission and further operations (1-3). In addition to
medical problems, increased surgical costs are an additional
concern. An American study reported a total cost of $1.3
billion annually for the treatment of postoperative peritoneal adhesions (4).
Peritoneal adhesions that occur following surgical trauma
are caused by metabolic processes and the combination of a

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

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CLINICS 2012;67(11):1303-1308

A laparotomy was performed via a 3 cm midline incision.
The cecum was pulled from the abdomen and scrubbed five
times with a sterile toothbrush to induce a subserosal
hemorrhage on an area that was equivalent to the toothbrush surface. The cecum was then returned to its normal
position.
Prior to closing the abdomen, 3 mL of 0.9% saline solution
(Eczacibasi-Baxter, Istanbul-Turkey), 3 mL of 4% icodextrin
(ADEPTTM, Baxter, Deerfield, USA), or 3 mL of canola oil
(YudumTM Canola Oil, Balikesir-Turkey), which was sterilized in an autoclave and cultured prior to use, were
intraperitoneally administered to the three groups of rats
(n = 8 each).
To overcome fluid leakage from the peritoneal cavity, the
wound edges were held together by four clamps immediately
following the application of the protocol material. The
abdominal incision was subsequently closed with 4-0 polypropylene running sutures (ProleneTM, Ethicon, Cornelia, GA,
USA). Then, 100 mL/kg of paracetamol (PerfalganTM, Bristol
Myers Squibb, Park Avenue, NY, USA) was injected
subcutaneously for analgesia. Normal feeding was allowed
after 6 hours. Wound healing and abdominal wall integrity
were assessed daily over the first three days following the
surgery. All of the rats were sacrificed 10 days postoperatively
using a high dose (100-150 mg/kg) of sodium thiopental
(PentotalTM, Abott, Illinois, USA).

alpha (1-4) and alpha (1-6 [,10%]) glucosidal bonds. When
administered intraperitoneally in a 4% solution, icodextrin
functions as a colloidal osmotic agent. The colloidal osmotic
action of this polymer retains a reservoir of fluid within
the peritoneal cavity for 3-4 days. Icodextrin provides a
temporary physical separation of the peritoneal surfaces by
hydroflotation as the result of maintaining a fluid reservoir.
This effect minimizes tissue apposition during the critical
period of fibrin formation and mesothelial regeneration
following surgery, thereby providing a barrier to adhesion
formation (13).
Canola oil, which is also referred to as low erucic acid
rapeseed oil, is a vegetable oil that contains monounsaturated
fatty acids, oleic acid (55%), and polyunsaturated fatty acids
(PUFAs), which are composed of linoleic acid (v-6) (25%) and
alpha-linoleic acid (v-3) (10%). Canola oil has the lowest
concentration of saturated fatty acids (SFA, 4%) of all the
commonly consumed oils, and it is a good source of vitamins
E and K and phytosterols (14). Although erucic acid is a
monounsaturated fatty acid and a member of the v-9 FA
family, it metabolizes to oleic acid and has anti-inflammatory
effects (15). V-3 PUFAs are essential nutrients that play a
beneficial role in several disease processes because of their
anti-inflammatory, analgesic, anti-thrombotic, and antimutagenic effects. These fatty acids also modulate some
forms of lipids and positively affect the central nervous
system. In contrast, v-6 fatty acids have inflammatory,
nociceptive, thrombotic, and mutagenic effects.
Canola oil is a lipid that can separate the traumatic
peritoneal surfaces, and it contains fatty acids that can
inhibit adhesion formation; therefore, it may successfully
prevent peritoneal adhesions. Thus, the aim of this experimental study is to compare the macroscopic, histopathological and biochemical effects of icodextrin and canola oil in
preventing postoperative peritoneal adhesions.

Macroscopic assessment
The peritoneal cavity was entered via a ‘‘reverse U’’
incision without damaging the formed adhesions. Retracting
the anterior abdominal wall caudally, the peritoneal cavity,
the small bowels and the cecum were carefully inspected and
assessed according to the Blauer staging scale (16) (Table 1).
Following a macroscopic evaluation, a 2-cm ileocecal
segment and its neighboring mesenteric root (0.560.5 cm)
were resected for the histopathological and biochemical
examinations. The sacrificed animals were placed in the
Marmara University Experimental Animal Laboratory’s
medical waste, and the study was completed.

MATERIALS AND METHODS
This study was performed at the Experimental Animal
Laboratory of Marmara University Medical Faculty after
obtaining approval from the Animal Ethics Committee. All
of the protocols followed the declaration of Helsinki
guidelines concerning the care and use of laboratory
animals.
Twenty-four Wistar albino outbred female rats (mean
weight 250¡30 g, mean age seven months) were divided
into three groups and were housed in standard rat cages,
each containing a maximum of five rats. The rats were
housed using a 12-hr light/12-hr dark cycle at stable
temperatures (between 19 and 22 ˚C). The animals were
provided with standard rat pellet and tap water ad libitum.

Histopathological assessment
The resected adhesion model specimen was fixed in a
10% formalin solution. Following dehydration, the samples
were paraffin-embedded using tissue processing equipment
(Leica ASP300S, Newcastle-UK). Four 3 mm-thick slices from
each intestinal segment were obtained using a microtome
(Leica RM2255, Newcastle-UK).
As defined by the producers of the standard protocol, the
first section was stained with hematoxylin and eosin (HE)
(Bio optica, Milano-Italy, Bio stain, Manchester-UK) to
assess giant cell, lymphocyte/plasmocyte, neutrophil, and
histiocyte reactions. The second section was stained with
Masson Trichrome (Sigma Aldrich, St. Louis, MO-ABD) to
assess fibrosis. The third section was stained for CD54/
ICAM1 (NovocastraTM Leica, Newcastle-UK, 23G12 clone)
and CD31/PECAM1 (NovocastraTM Leica, Newcastle-UK,
1A10 clone).
The slides that were stained with HE to assess the giant
cell, lymphocyte/plasmocyte, neutrophil, and histiocyte
reactions, as well as the slides that were stained with
Masson Trichrome for the fibrosis analysis, were scored
from zero to three based on the criteria that were used by
Delaco et al. (17). The two slides that were stained for CD54
and CD31 were also stratified from zero to three, as shown

Operation and adhesion model
Following a 12-hour starvation, the rats were anaesthetized with IM ketamine hydrochloride (KetalarTM,
Eczacıbas¸ı, Istanbul-Turkey) at 40 mg/kg and xylazine
(ROMPUNTM, Bayer, Berlin Germany) at 10 mg/kg body
weight. The rats were placed in a supine position, and their
extremities were affixed to the operating table with plaster.
All of the operations were performed using powder-free,
non-latex gloves to prevent the anticipated peritoneal
adhesions caused by foreign body reactions. After abdominal skin shaving, antisepsis was maintained using povidone iodine (BetadineTM, Kurtsan, Istanbul-Turkey).

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Table 1 - The scoring system used for the macroscopic and microscopic evaluations of the inflammatory reactions on
serosal surfaces (16).
SCORE
0
1
2
3
4
*

Macroscopic findings

Cellular reaction*

Fibrosis

ICAM1 and PECAM1 staining

No adhesions
Thin and narrow, easily separable
adhesions
Thick adhesions, limited to one area
Thick and wide adhesions
Thick and wide adhesions between the
organs and the abdominal wall

in Table 1. The microscopic assessment was made using
light microscopy (Nikon E600, Tokyo-Japan) under 100x
and 200x magnifications. Images were obtained of the
observed samples (Nikon E5400, Tokyo-Japan). The pathologist was blinded to the study group.

with a confidence interval of 95%, and p-values below 0.05
were considered to be statistically significant.

RESULTS
No postoperative complications, such as bowel obstructions and peritonitis, or mortality was observed in any of the
groups during the study. At necropsy assessment, no
intraperitoneal fluid was found at day 10.

Biochemical assessment
The mesenteric tissue samples that were used for the
biochemical assessment were kept in dry tubes and taken to
the biochemistry laboratory. After collecting all of the
tissues, 70 mg of tissue were homogenized in a 1 mL 0.9%
NaCl solution (Janke & Kunkel Ultra-Turrax T25, StaufenGermany). After adding equal volumes of HCl, the
homogenized tissues were incubated for 24 hours in a
95 ˚C water bath.
The compounds that were used for the analysis, i.e.,
acetate citrate buffer (pH 6.5), chloramine T and Erlich
reactive, were all purchased from Sigma Aldrich St. Louis,
MO, USA, and were freshly prepared. A standard hydroxyproline study was prepared. After completing the study,
the absorbency values from the samples and the standards
were quantified using a spectrophotometer (Beckman
Coulter DU-530, Brea, CA, USA).

Macroscopic assessment
The rats in both the canola and the icodextrin groups,
nearly half of which were free from adhesions, appeared to
have lower macroscopic scores than those in the control
group. However, this difference was not statistically
significant (p = 0.17) (Table 2, Figure 1).

Histopathological assessment
The microscopic assessment of the giant cell (p = 0.381),
lymphocyte/plasmocyte (p = 0.126), and neutrophil reactions (p = 0.307) revealed no statistically significant difference in comparison between the control, icodextrin and
canola groups.
Although no histiocytic reaction was observed in the
sham or the icodextrin-treated groups, a significant histiocytic reaction was observed in the rats in the canola oiltreated group: two rats had scores of zero; three rats had
scores of one; and three rats had scores of two (p = 0.001)
(Table 2, Figure 1).
The fibrosis assessment revealed that both the icodextrinand the canola oil-treated groups exhibited significantly
decreased fibrotic reactions compared to the control group;
none of the icodextrin- or canola-treated rats received a
score of three (p = 0.025) (Table 2, Figure 1).
The immunohistochemistry studies revealed that the rats
in the control and icodextrin groups expressed higher ICAM
and PECAM1 levels in the cellular membranes: the mean

Statistical analyses
All of the statistical analyses were performed using the
SPSS statistical software package (version 16.0, IBM, USA).
The numerical data were expressed as the mean and
standard derivation, unless otherwise stated. Because the
values were nonparametric and the number of rats in the
groups was under 30, the Kruskal–Wallis test was used for
the statistical analysis of the giant cell, lymphocyte/plasmocyte, neutrophil, and histiocyte reactions and for the fibrosis,
ICAM1 and PECAM1 scores. The statistical significance of
the hydroxyproline levels was assessed using an ANOVA
test given that all of the values that were obtained from the
three groups were parametric. The results were evaluated

ICAM scores were 1.25¡0.70, 1.75¡1.04 and 0.88¡0.64 for
the control, icodextrin-treated and canola oil-treated groups,
respectively; the mean PECAM1 scores were 1.12¡0.83,
1.38¡0.74 and 0.54¡0.50 for the control, icodextrin-treated
and canola oil-treated groups, respectively. However, there
was no statistically significant difference between the three
groups (p = 0.14 and p = 0.069) (Table 2, Figure 1).

DISCUSSION
Postoperative peritoneal adhesions can lead to readmissions and reoperations caused by mechanical small
bowel obstruction, which can increase clinical workloads
and hospital costs. Many preventive measures have been

Biochemical assessment
The mean hydroxyproline levels were 302.65¡
147.69, 273.40¡118.56 and 151.14¡53.30 in the control,

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system. These cell types are the most important chronic
inflammation-mediating cells with respect to the subsequent
response of other immune cells and the cellular mediators to
injury. A fibrin matrix is exuded from inflammatory cells,
and this matrix is gradually organized into fibrin bands that
contain fibroblasts, macrophages and giant cells that bridge
the two injured peritoneal surfaces (33). Several animal
studies have revealed the cellular response to peritoneal
damage, and many therapeutics have been designed to
prevent adhesion formation by interacting with the extracellular matrix and cellular mediators. Thus, it appears that
the complex relationship between peritoneal healing and
adhesion formation will continue to be investigated until the
pathogenesis of adhesions is completely understood.
In certain studies, polyunsaturated fatty acids and their
derivatives, eicosapentaenoic acid and docosahexaenoic
acid, have been observed to promote peritoneal wound
healing by activating the inflammatory cascade via peroxisome proliferator activated receptors (PPARs). This activation, which mediates lipid metabolism, fatty acid oxidation,
and cytokine production, can induce the anti-adhesogenic
effects of v-3 fatty acids. This effect is mediated by reducing
the levels of type 1 collagen, vascular endothelial growth
factor, and transforming growth factor b-1 (34). This
tremendous modulation of the inflammatory response
indicates the potential anti-adhesive effects of PUFAs.
However, whether this action is beneficial or detrimental
in clinical wound healing is unknown.
PUFAs affect numerous physiological processes that
modulate the physical properties of the lipid bilayer
composition and the fluidity of the cell membrane. Our
study focused on the cellular response to peritoneal damage
and revealed a significant decrease in fibrosis in the canola
oil-treated group compared to the icodextrin-treated and
control groups. Although both icodextrin and canola oil
reduced adhesions macroscopically, this alteration did not
reach statistical significance. Meanwhile, cellular reactions,
such as giant cells, mononuclear and polymorph nuclear
leukocyte reactions, did not differ in any of the groups. This
result may be explained as the neutral effect of both of the
tested anti-adhesion materials on normal wound healing. In
contrast, a very prominent histiocytic reaction was observed
in the canola oil-treated group (1.12ÂĄ0.84), whereas no such
reaction was observed in the other two groups. This result
suggests that tissue macrophages also contributed to the
inflammatory process in animals following canola oil
treatment.
Soybean oil, which contains linoleic acid (51%), oleic acid
(25%), methyl methacrylate, palmitic acid, linolenic acid,
and stearic acid, have been experimentally tested for their
abilities to decrease the severity of postoperative peritoneal
adhesions and have been reported to decrease adhesion
formation when applied prior to the peritoneal trauma (6).
Vitamin E, when applied just prior to the closing of the
incision, was effective in reducing adhesion formation (8). In
agreement with this previous study, Durmus et al. demonstrated that vitamin E and selenium, which are believed to
be commonly used antioxidants, thoroughly decreased
fibrosis and intra-abdominal adhesions by reducing hydrogen peroxides and lipid hydroperoxides to nontoxic
elements (35). Canola oil, a good source of vitamin E, may
also prevent peritoneal adhesions.
The fact that no dose adjustments were performed in the
canola oil-treated group is an important limitation of the

proposed to overcome this problem, including meticulous
surgical techniques, excellent intraoperative hemostasis,
avoiding unnecessary handling of the bowel, creating
devitalized or ischemic tissue, minimizing the risk of foreign
body granulomas (primarily surgical glove powder and
excessive suture material), and preventing peritoneal contamination; even certain serosal plication techniques have
been proposed to overcome this problem (18).
In addition to these techniques, a wide range of
biologically active substances in the form of simple fluids,
gels and solids, either combined or alone, have been
investigated both clinically and experimentally to reduce
or prevent abdominal adhesions (19). These novel
approaches aim to prevent adhesion formation by physically separating the surgically manipulated areas via
irrigation and instillation or by covering the serosal surfaces
with liquid agents or barriers in the form of films, sprays or
gels. Carboxymethyl cellulose + hyaluronic acid is the most
examined material (20-26).
Oxidized regenerated cellulose and polytetrafluoroethylene are two other synthetic absorbable barriers that have
been shown to effectively reduce the incidence of surgical
adhesions (27).
An ideal barrier should be biodegradable, biocompatible
and surgically easy to handle and should act locally to avoid
side effects. However, only certain materials meet all of
these requirements, and no large prospective, randomized
double-blind human studies have demonstrated their
efficacy. Furthermore, none of these materials have been
widely adopted by surgeons, indicating that the materials
only decrease adhesion severity, not incidence (28,29).
The effect of a 4% icodextrin solution, which has received
limited approval by the FDA for use in laparoscopic
gynecological surgery, was largely investigated using
ARIEL registry data. These results indicated that this
solution was widely accepted by both gynecological
surgeons and patients (30). This clinical evaluation had
been conducted in patients who underwent a routine
gynecological surgery via either laparoscopy or laparotomy
in six European countries. The participating surgeons were
asked to use 4% icodextrin solutions for the lavage and
instillation of the peritoneal cavity. A questionnaire was
used to assess the patientsâ&#x20AC;&#x2122; experiences, and it revealed a
high acceptability rate of this method, with low rates of
adverse events (7.5% and 13.9% of the patients who
underwent laparoscopy and laparotomy, respectively) (31).
The same group achieved similar results in general surgery
patients but observed a substantial number of adverse
effects (16.7% and 30.6% of the patients who underwent
laparoscopy and laparotomy, respectively) (32). Nevertheless, in the context of laparoscopic pelvic surgery, the
use of this solution is avoided in bowel resections and
peritoneal inflammatory conditions, which may require
more robust adhesion prevention techniques.
It is believed that other agents and pharmaceutics in the
form of gels, sprays or liquids might be rapidly absorbed by
the peritoneum. Studies of these compounds, which were
performed primarily in animals, demonstrated conflicting
results (5-12,17). Thus, more evidence is needed.
The mechanism of peritoneal healing is similar to wound
healing. However, regardless of the size of the peritoneal
trauma, healing requires approximately 7-10 days. Peritoneal
leukocytes, histiocytes, and tissue-consolidating mature
macrophages are components of the monocyte-phagocytic

present study. The effect of intraperitoneally administering
canola oil has, to our knowledge, never been examined.
Further investigations are required to determine the most
effective dose and form in which canola oil can optimally
prevent adhesions.
Given the benefits of canola oil in inducing the histiocytic
reaction and lowering hydroxyproline levels, the data that
are presented here demonstrate that intraperitoneally
administered canola oil decreases collagen synthesis and
has no detrimental effect on the wound healing process.
Compared with icodextrin, canola oil may be a promising
agent in the prophylaxis of adhesion formation.

AUTHOR CONTRIBUTIONS
Yigitler C contributed to the hypothesis, study design, data collection,
assessment of the results and manuscript preparation. Karakas DO
contributed to the study design, animal experimentation, data collection
and assessment. Kucukodacı Z contributed to the histopathological
evaluation. Cosar A contributed to the biochemical tests and evaluations.
Gu¨lec B contributed to the data assessment and manuscript preparation.
Akin contributed to the hypothesis and study design.

OBJECTIVE: This study evaluated the performance of lungs that were preserved with different solutions (Celsior,
Perfadex or saline) in an ex vivo rat lung perfusion system.
METHODS: Sixty Wistar rats were anesthetized, anticoagulated and randomized into three groups (n = 20). The rats
were subjected to antegrade perfusion via the pulmonary artery with Perfadex, Celsior, or saline, followed by 6 or
12 hours of ischemia (4 ˚C, n = 10 in each group). Respiratory mechanics, gas exchange and hemodynamics were
measured at 10-minute intervals during the reperfusion of heart-lung blocks in an ex vivo system (IL2-Isolated
Perfused Rat or Guinea Pig Lung System, Harvard Apparatus, Holliston, Massachusetts, USA; Hugo Sachs Elektronik,
Germany) for 60 minutes. The lungs were prepared for histopathology and evaluated for edema following
reperfusion. Group comparisons were performed using ANOVA and the Kruskal-Wallis test with a 5% level of
significance.
RESULTS: Gas exchange was not significantly different between lungs perfused with either Perfadex or Celsior at the same
ischemic times, but it was very low in lungs that were preserved with saline. Airway resistance was greater in the lungs that
were preserved for 12 hours. Celsior lungs that were preserved for 6 and 12 hours exhibited lower airway resistance
(p = 0.01) compared to Perfadex lungs. Pulmonary artery pressure was not different between the groups, and no significant
differences in histopathology and apoptosis were observed between the groups.
CONCLUSIONS: Lungs that were preserved with Celsior or Perfadex exhibited similar gas exchange and
histopathological findings. Airway resistance was slightly lower in the Celsior-preserved lungs compared with the
Perfadex-preserved lungs.
KEYWORDS: Lung Transplantation; Organ Preservation Solutions; Animal Experiment.
Menezes AQ, Peˆgo-Fernandes P, Cardoso PF, Braga KA, Nepomuceno NA, Pazetti R, et al. Comparison of Celsior and Perfadex lung preservation
solutions in rat lungs subjected to 6 and 12 hours of ischemia using an ex-vivo lung perfusion system. Clinics. 2012;67(11):1309-1314.
Received for publication on May 31, 2012; First review completed on June 26, 2012; Accepted for publication on July 31, 2012
E-mail: paulo.fernandes@incor.usp.br
Tel.: 55 11 2661-5248

on several factors, including the quality of lung preservation, which is important in the early stages after transplantation (2).
Lung preservation for transplantation enables distant
organ procurement, objectively improves organ quality after
reperfusion and decreases post-ischemic reperfusion injury.
The most widely used preservation method is hypothermia
with the administration of preservation solutions with or
without pulmonary vasodilators (3,4).
Low-potassium preservation solutions are classified as
extracellular-type solutions and include Perfadex and
Celsior, which are most commonly used for lung preservation. The Celsior solution was originally developed for
heart preservation (2), and Perfadex was developed for lung
preservation (5).

INTRODUCTION
Lung transplantation is a well-established treatment for
end-stage lung disease. However, lung transplant-related
mortality remains significant despite the increasing number
of transplants that are performed (over 3,200 in 2010) (1).
Ischemia-reperfusion injury is the primary cause of lung
transplant-related mortality. The severity of injury depends

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

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CLINICS 2012;67(11):1309-1314

Ventilation was initiated with the heart-lung block positioned in the ex vivo perfusion system (RR = 60 bpm;
inspiratory/expiratory ratio = 60%; one breath/minute = 50%
increase in tidal volume; and PEEP = 1 cmH2O), beginning at
50% of the tidal volume and increasing until a ventilation of
10 mL/kg body weight was reached. Perfusion of the block
began at a low flow rate (2 mL/min) and was increased over
5-10 minutes until the desired flow rate (5-7 mL/min) was
reached. Ventilation and perfusion were stabilized, and data
(hemodynamics, ventilatory mechanics, and pulmonary
arterial and venous gases) were collected every 10 minutes
for 60 minutes.
Blood samples were taken from the pulmonary arterial
and left atrial cannulae for blood gas measurements (ABL
800, Radiometer, Denmark). The lung’s relative oxygenation
capacity (ROC) was calculated using the following formula:
ROC = [(PvO2-PaO2) 6100]/PaO2 (7). The PaO2 corresponds
to the deoxygenated blood that was taken from the
pulmonary arterial cannula, and the PvO2 is the oxygenated
blood from the left atrial cannula. The pH of the blood in the
reservoir was corrected with sodium bicarbonate (0.3 mEq/
L/dose) to maintain the pH between 7.1-7.4 (9,10). The
ventilatory mechanics parameters were provided by the IL2
system and included tidal volume, lung compliance and
maximum inspiratory and expiratory flows. The hemodynamic parameters included pulmonary artery pressure and
pulmonary resistance.
The left lung was dissected out at the end of perfusion.
The lung was weighed, stored at 70 ˚C for 72 hours, and reweighed for the calculation of the wet-to-dry (W/D) weight
ratio.
The right lung was immersed in 10% buffered neutral
formalin solution for 24 hours and then longitudinally
sectioned. The most volumetrically significant half was
selected for embedding and processing. The specimen
was dehydrated, diaphanized and embedded in paraffin.
Histological sections (5 mm thickness) were stained with
hematoxylin and eosin (HE) for light microscopy analysis.
Histopathological analyses of the fragments was performed
to assess the presence or absence of congestion, alveolar
edema, bleeding (alveolar and/or interstitial), acute thrombosis, interstitial inflammatory infiltrates (mononuclear
and/or granulocytic) and pneumonic foci.
Fragments were obtained from the right lung immediately after reperfusion for the detection and quantification of
apoptotic cells using the In situ Cell Death Detection
Kit (Roche, Mannheim, Germany). The fragments were
immersed in 10% buffered formalin for fixation for
24 hours. Paraffin blocks of the specimens were prepared
and sectioned into slices of 5 mm thick sections. The sections
were deparaffinized in three xylene baths (five minutes
each) and rehydrated in an ethanol gradient (100, 95, 90, 80,
and 70%). Proteinase K was applied to the sections for 30
minutes at room temperature. The sections were washed in
two (three minutes each) PBS (phosphate-buffered saline)
baths. The sections were incubated in a 0.3% hydrogen
peroxide (H2O2) and methanol solution for 30 minutes at
room temperature and immediately washed twice in PBS. A
TUNEL mixture (50 ml:5 ml of the TdT enzyme solution and
45 ml of the labeled nucleotide solution) was applied to each
specimen. The specimens remained in the wet chamber at
37 ˚C for 60 minutes. The sections were washed three times
in PBS and coverslipped with glycerin for fluorescence
microscopy analysis.

Perfadex was gradually introduced after its original
development two decades ago, despite the superiority of
the experimental results obtained from this solution
compared with other solutions (2). The use of preservation
solutions in the lungs reduces the incidence of acute graft
failure after transplantation from 30% to less than 15% (2).
Controversy exists over the benefits of Perfadex based on its
late clinical performance and associated 1-year post-transplant mortality rate (2). The recent introduction of ex vivo
lung reconditioning and donation after cardiocirculatory
arrest has rekindled this controversy and reinforced the
need for the reassessment of the currently used preservation
solutions (6).
Ex vivo lung perfusion systems are useful for experimental physiological evaluations because of its reproducibility and relatively low cost for assessments of lung
preservation methods (7,8).
This study compared the functional performance of rat
lungs that were subjected to different ischemic times;
preserved with Perfadex, Celsior or normal saline; and
reperfused in an ex vivo lung perfusion system.

MATERIALS AND METHODS
Animals were handled in accordance with the Guide for
the Care and Use of Laboratory Animals prepared by the
Institute of Laboratory Animal Research and published by
the National Academies Press, 8th Edition, 2011.
Male Wistar rats (250 to 300 g) were anesthetized with
sodium thiopental (50 mg/kg, intraperitoneally) and subjected
to sternolaparotomy, tracheostomy, and mechanical ventilation
(room air; tidal volume (Vt) = 10 mL/kg body weight; respiratory rate (RR) = 70 cycles/min; and positive end expiratory
pressure (PEEP) = 1 cmH2O). The diaphragm was opened
radially to expose the supradiaphragmatic vena cava after
anticoagulation (heparin 1,500 IU via the inferior vena cava).
The right ventricle outflow tract was incised adjacent to the
pulmonary artery, the inferior vena cava and the left ventricle.
The pulmonary artery was cannulated through right ventriculotomy, and the lungs were perfused antegradely with 20 mL of
a hypothermic solution (4˚C) at a pressure of 10 cm H2O, which
was obtained by the elevation of the reservoir. The effluent was
drained via the left ventriculotomy. Animals were randomized
into three groups (n = 20 each) based on the perfused
preservation solution: PerfadexH (Vitrolife, Kungsbacka,
Sweden), CelsiorH (Genzyme, Catalent Limoges S.A.S.,
France) or a 0.9% saline solution (Baxter, Sa˜o Paulo, Brazil).
The heart-lung block was removed partially inflated at the end
of perfusion, immersed in the perfusion solution and stored at
4-7˚C for 6 or 12 hours according to group assignment. The
animals were divided into two groups (n = 30) according to
ischemic time (6 or 12 hours), and each group was subdivided
into three groups (n = 10) according to perfusion solution.
The heart-lung block was connected to the ex vivo
perfusion system at the end of the cold ischemia period
(IL-2 Isolated Perfused Rat or Guinea Pig Lung System;
Harvard Apparatus, Holliston, MA, USA; Hugo Sachs
Elektronic, Hugstetten, Germany) and reperfused for 60
minutes using homologous blood from donor rats diluted in
saline to a hematocrit of 15-20%. The pulmonary venous
blood was deoxygenated using a membrane oxygenator (D150 Hemofilter, Medsulfone, Italy) that contained a continuously administered (100 mL/min flow rate) gas mixture
(90% N2 and 10% CO2).

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Lung preservation: Celsior vs. Perfadex
Menezes AQ et al.

Figure 1 - Mean relative oxygenation capacity (ROC) of rat lungs subjected to ischemia and reperfusion for 60 minutes, illustrating no
significant differences between Celsior- and Perfadex-perfused lungs at either ischemic time. The lungs subjected to 6 hours of cold
ischemia exhibited higher ROCs than the 12-hour ischemic lungs, but this difference was significant only for the lungs preserved with
saline (p = 0.001).

Small fragments of the right lung (approximately 2 mm
62 mm) were used for transmission electron microscopy
analysis. The material was placed in universal fixative (1%
glutaraldehyde, 1% paraformaldehyde, pH = 7.4) immediately after biopsy, postfixed in osmium tetroxide (2%),
dehydrated, and embedded in epon-araldite. Slices (1 mm
thick) were analyzed using a transmission electron microscope for the qualitative evaluation of apoptotic changes in
type II pneumocytes.

this difference did not reach statistical significance (6 hours,
p = 0.75; 12 hours, p = 0.18). Oxygenation was lowest in the
lungs that were preserved with saline for 12 hours
(p = 0.001). However, no significant differences between
Celsior and Perfadex lungs were observed at either ischemic
time (Perfadex, p = 0.06; Celsior, p = 0.17) (Figure 1).
The compliance of the lungs that were preserved with
Celsior was superior to that of the saline lungs at 6 hours
(p = 0.03) but not at 12 hours (p = 0.07). However, lung
compliance was not different between the Celsior and
Perfadex lungs at either ischemic time (6 hours, p = 0.16;
12 hours, p = 0.31). Overall, the compliance of the 6-hour
lungs was greater than that of the 12-hour lungs (Perfadex,
p = 0.02; Celsior, p = 0.01; saline, p = 0.01) (Figure 2).
The Celsior lungs exhibited the lowest airway resistance
for both ischemic times (p = 0.01). The Perfadex-preserved
lungs exhibited lower pulmonary resistance than the salinepreserved lungs at 6 hours (p = 0.03), but this difference was
not observed at 12 hours (p = 0.16). Airway resistance was
higher in the 12-hour ischemic lungs regardless of the
preservation solution (Perfadex, p = 0.001; Celsior, p = 0.003;
saline, p = 0.006) (Figure 3).

Statistical analysis
Statistical analyses included the parametric ANOVA and
nonparametric Kruskal-Wallis tests. The chi-square test was
employed for qualitative variables. The data are presented
as the means and standard errors of the mean, and the
significance level in this study was 5% (p,0.05). Descriptive
and inferential statistical analyses were performed with
SPSS software version 13 (SPSS 13.0 for Windows).

RESULTS
The ROC of Celsior lungs was superior to that of Perfadex
or saline lungs, regardless of the ischemic time. However,

Figure 2 - Mean lung compliance of rat lungs submitted to ischemia and reperfusion for 60 minutes, illustrating no significant differences
between lungs preserved with Celsior and Perfadex. Lungs subjected to ischemia for 6 hours performed better than those subjected to
12 hours of ischemia.

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CLINICS 2012;67(11):1309-1314

Figure 3 - Airway resistance of rat lungs subjected to ischemia and reperfusion for 60 minutes. The Celsior lungs exhibited the lowest
airway resistance for both ischemic times. The airway resistance was lower in lungs submitted to 6 hours of ischemia compared with
those submitted to 12 hours of ischemia.

The W/D ratio was not different between the 6- and 12-hour
lungs regardless of the preservation solution (p = 0.29 and
p = 0.26, respectively). The Perfadex-preserved lungs exhibited
a higher W/D ratio at 12 hours of ischemia compared with
6 hours (Perfadex, p = 0.001; Celsior, p = 0.27; saline, p = 0.13).
Pulmonary artery pressure was not different between the 6and 12-hour lungs regardless of the preservation solution
(p = 0.88 and p = 0.98, respectively), and no significant difference between lungs in the two ischemic time groups was
observed (Perfadex, p = 0.17; Celsior, p = 0.34; saline, p = 0.19).
The saline-preserved lungs exhibited the highest levels of
alveolar edema on light microscopy regardless of the
ischemic time (6 hours, p = 0.006; 12 hours, p = 0.001). The
Celsior and Perfadex lungs exhibited some level of alveolar
edema at both ischemic times (6 hours, p = 0.131; 12 hours,
p = 1.00), but no significant differences in edema formation
across groups and between 6- and 12-hour ischemic lungs
were observed (Perfadex, p = 0.37; Celsior, p = 0.3; saline,
p = 0.47) (Figure 4).
No differences in type II pneumocyte changes were
observed on electron microscopy between the different groups
and between the 6- and 12-hour ischemic lungs. Very few
epithelial cells exhibited gross chromatin aggregates peripheral to the nucleus, which is indicative of the initial stages of
apoptosis. A slight thickening of the basement membrane due
to edema was observed in all groups (Figure 5).
The TUNEL assay for the assessment of apoptosis did not
demonstrate any statistically significant differences between
groups, regardless of the preservation solution and ischemic
time (Figure 6).

Perfadex or Celsior. Both Perfadex and Celsior are extracellular solutions, but the potassium content of the Perfadex
solution is lower than that of the Celsior solution (4 vs.
15 mEq/L, respectively). Therefore, Perfadex is potentially
less harmful to the structural and functional integrity of
endothelial cells and may, as a result, decrease the production of oxidants and vasoconstrictors. The addition of
Dextran-40 increases the oncotic pressure, which improves
the deforming capacity of red blood cells, prevents erythrocyte aggregation and induces disaggregation of the aggregated red blood cells. Therefore, Dextran-40 elicits an

This study demonstrated a similar reperfusion performance of gas exchange in lungs that were preserved with

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CLINICS 2012;67(11):1309-1314

Lung preservation: Celsior vs. Perfadex
Menezes AQ et al.

Similar lung compliance results were found in this study
between the Celsior and Perfadex groups for both ischemic
times. The Celsior solution exhibited a tendency for
improved lung compliance during reperfusion, but this
difference was not significant. Sommer et al. observed
comparable lung compliances in a pig model in both
experimental groups after 24 hours of cold ischemia and
7 hours of reperfusion (14). Wittwer et al. also described
that dynamic lung compliance in a pig model remained
stable over 6 hours of reperfusion even after 27 hours of
hypothermic ischemia. These authors did not demonstrate a
significant difference between solutions regardless of the
perfusion route (antegrade or retrograde). However, preservation with Celsior produced the lowest lung compliance
values across the investigated groups (15). Both preservation solutions in our study were associated with significantly lower values of lung compliance after 12 hours of
ischemia, which suggested that this parameter was negatively affected by longer ischemic times regardless of the
preservation solution.
Celsior performed better than Perfadex with regard to
airway resistance, as demonstrated by the lower resistances
measured during the reperfusion of the Celsior lungs.
Increased resistance may be secondary to the increases in
permeability and alveolar-capillary injury that result from
edema. However, airway resistance may vary due to the
denervation of the heart-lung block and to the airway
reactivity, which is less likely to play a role in this model.
However, the microscopic observations of alveolar edema
were similar in the Perfadex and Celsior lungs despite the
significant differences in airway resistance caused by these
two solutions.
Our experimental design included a saline solution group
to assess the stability and reliability of the model. The
absence of significant differences in oxygenation and
pulmonary resistance parameters between Perfadex and
saline lungs may be partly attributed to blood dilution.
Puskas et al described these effects in a similar ex vivo
perfusion model in which blood dilution with a crystalloid
solution significantly improved post-ischemic reperfusion
injury (2). The blood dilution may have been higher in the
saline group in the present model because the vascular bed
of the lungs was filled with saline prior to reperfusion. This
act may explain the lower pulmonary resistance values in
the saline-preserved lungs compared with the Perfadex
lungs for the 12-hour ischemic period. This act may also
underlie the biases that are inherent in the absence of the
body of the animal and its replacement with a deoxygenator. The use of Krebs solution instead of saline may be an
option for preventing the aforementioned effects of blood
dilution.
Wittwer et al. (12) also observed no significant differences
in pulmonary artery pressure between Perfadex- and
Celsior-preserved lungs using a similar model. One limitation of this model is that it does not enable the calculation of
pulmonary vascular resistance, which yields a better
assessment of pulmonary hemodynamics. These authors
used a swine model and pulmonary vascular resistance
calculations to conclude that preservation with Celsior
produced the highest resistance values, but Sommer et al.
(14) reported that Perfadex was inferior to Celsior.
The ROC is the parameter of choice for the assessment of
gas exchange in this ex vivo model and likely represents the
most important physiological parameter (7). We did not

antithrombotic effect due to its action on the surfaces of
endothelial cells and platelets. These effects improve lung
microcirculation and preserve the endothelial-epithelial
interface, which can reduce water and protein extravasation
during reperfusion (2).
Previous studies on the use of Celsior or Perfadex for lung
preservation reported comparable results, but these studies
used shorter ischemic times (7). The Celsior solution
contains antioxidant substances, such as histidine, mannitol,
lactobionate, and glutathione (11).
All lungs that were subjected to 6 or 12 hours of ischemia
in the present study completed the 60-minute reperfusion
period and provided consistent data for analysis. Other
authors have used ischemic times of 2 and 4 hours using the
same model used in the present study (7,12).
The ex vivo lung perfusion model is suitable for initial
evaluations of lung preservation because of its simplicity,
reproducibility, reliability, and low cost compared with
larger animal models (13).

observe a significant difference in the gas exchange performance of Celsior lungs compared with Perfadex lungs at either
ischemic timepoint examined. A previous study using a similar
model with 4 hours of ischemia demonstrated differences
between the two solutions that favored Celsior (7), but the
ischemic time in the present study is three times longer.
However, the saline solution group exhibited a decline in ROC
for the longest ischemic time.
The W/D ratios demonstrated that the increased edema
during reperfusion was closely related to the ischemic time.
Increased edema results from post-ischemic reperfusion
injury and the characteristics of the ex vivo model, which
uses an extracorporeal circuit, inorganic interfaces and nonpulsatile flow. The Perfadex-preserved lungs in the 6-hour
ischemic group exhibited a trend toward a lower weight
gain than the Celsior-preserved lungs, but this difference
was not statistically significant. The Perfadex solution is
associated with less edema regardless of the perfusion route
(15). An assessment of lung edema in pigs demonstrated
that Perfadex better prevents the formation of intra-alveolar,
peribronchovascular and septal edema and injury to the
alveolar-capillary barrier during ischemia-reperfusion than
Celsior (16). Another study demonstrated that the water
content of Perfadex lungs was not different than that of
Celsior lungs after 24 hours of cold ischemia and 7 hours of
reperfusion in pig lungs (14). Conversely, our study
demonstrated that Celsior lungs subjected to 12 hours of
ischemia were less edematous at the end of reperfusion than
Perfadex lungs. Overall lung performance in the Celsior
lungs that were subjected to 12 hours of ischemia was
similar to the 6-hour ischemic lungs, thus suggesting that
the use of the Celsior solution produced less edema than the
Perfadex solution over longer ischemic times. Unfortunately,
such differences did not achieve statistical significance, and
the results should be confirmed in future studies.
In conclusion, this animal model of ex vivo lung perfusion
demonstrated that lungs preserved with Perfadex and
Celsior exhibited similar gas exchange, hemodynamics
and histopathological findings. The Perfadex-preserved
lungs subjected to 12 hours of ischemia were more
edematous, but Celsior-preserved lungs exhibited slightly
better ventilatory mechanics, as suggested by the lower
airway resistance. Future studies are required to confirm
these results and clarify the underlying mechanisms.

Canzian M performed the histopathological analysis. Santim JK executed
the experimental protocol. Jatene FB supervised all stages of the study.

ACKNOWLEDGMENTS
This study was performed as part of the Thoracic and Cardiovascular
Surgery Post-graduate Program, Heart Institute, Hospital das Clı´nicas, Sa˜o
Paulo University Medical School, Sa˜o Paulo, SP, Brazil. The study was
supported by grants from the Fundac¸a˜o de Amparo a` Pesquisa do Estado
de Sa˜o Paulo (FAPESP), Sa˜o Paulo, SP, Brazil.

AUTHOR CONTRIBUTIONS
Menezes AQ executed the experimental protocol, conducted the data
analysis and participated in the manuscript writing. Pego-Fernandes P
supervised all experimental stages and the manuscript preparation.
Cardoso PF executed the experimental protocol and participated in the
manuscript preparation. Braga KA and Nepomuceno NA executed the
experimental protocol and performed the data analysis. Pazetti R executed
the experimental protocol. Correia AT performed the statistical analysis.

To review all specific questionnaires regarding quality of life in osteoporosis and to describe their distinctive
indications, we searched Medline, the Scientific Electronic Library Online database, and the Latin-American and
Caribbean Health Sciences Literature database. Nine specific questionnaires related to osteoporosis quality of life
were found: 1) the Women’s Health Questionnaire, 2) Osteoporosis Quality of Life Questionnaire, 3) Osteoporosis
Assessment Questionnaire, 4) Osteoporosis Functional Disability Questionnaire, 5) Quality of Life Questionnaire of
the European Foundation for Osteoporosis, 6) Osteoporosis-Targeted Quality of Life Questionnaire, 7) Japanese
Osteoporosis Quality of Life Questionnaire, 8) the 16-item Assessment of Health-Related Quality of Life in
Osteoporosis, and 9) the Quality of Life Questionnaire in Osteoporosis (QUALIOSTTM). The Quality of Life
Questionnaire of the European Foundation for Osteoporosis is the osteoporosis-specific questionnaire most
commonly used in the literature. The Quality of Life Questionnaire of the European Foundation for Osteoporosis
and the Osteoporosis Quality of Life Questionnaire are targeted more toward fracture assessment, and the
Osteoporosis Functional Disability Questionnaire can be used for longitudinal studies involving exercise. In the
present study, the authors summarize all of the specific questionnaires for osteoporosis and demonstrate that these
questionnaires should be selected based on the objectives to be evaluated. Osteoporosis-specific quality of life
questionnaires should be validated in the language of the country of origin before being used.
KEYWORDS: Quality of Life; Osteoporosis; Questionnaires; QUALEFFO; OQLQ; OPAQ.
Madureira MM, Ciconelli RM, Pereira RM. Quality of life measurements in patients with osteoporosis and fractures. Clinics. 2012;67(11):1315-1320.
Received for publication on May 17, 2012; First review completed on June 15, 2012; Accepted for publication on July 10, 2012
E-mail: rosamariarp@yahoo.com / melisamadureira@uol.com.br
Tel.: 55 11 3061-7213

developing new treatments, particularly in chronic diseases
such as osteoporosis (4). Assessing quality of life in
osteoporosis is commonly used as an outcome measure
secondary to the biomechanical and radiographic evaluations following each fracture event (5,6).
Quality of life encompasses various facets of life,
including health status, environment, financial aspects and
human aspects. Health status is a subset of quality of life
that covers physical, mental, and social well-being (5,7).
To measure quality of life is to assess subjective feelings
objectively. Using quality-of-life questionnaires, we can
evaluate treatment effects in clinical trials (5,8,9). Questionnaires have been used in epidemiological studies to assess
quality of life and to obtain data regarding disease severity,
disease morbidity, health care, and treatment (5,6).
In this review, we describe the characteristics and specific
indications for osteoporosis quality of life questionnaires.
The purpose of the present article was to discuss the
adequacy of these questionnaires and their best indication
according to osteoporosis clinical studies.

INTRODUCTION
Defining quality of life
The World Health Organization Quality of Life (WHOQOL)
project was initiated in 1991. The aim of the project was to
develop an international cross-cultural quality of life assessment instrument. The WHOQOL instrument assesses individual perceptions in the context of culture, value systems,
personal goals, standards, and concerns (1). This definition
demonstrates that quality of life should not be measured by
the frequency with which a medical service is offered to the
patient but by the degree to which the results obtained serve
the purposes of prolonging life, easing pain, restoring
function, and preventing incapacity (2).
Two major developments in the health field are recognizing the importance of patient-based disease evaluations and
the importance of assessing the quality of the therapeutic
measures being employed (3).
Assessing functional status and quality of life has been
considered central to evaluating disease progression and

Quality of life and osteoporosis
Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.

Assessing health-related quality of life has been considered
an important marker of the clinical evolution of patients with
osteoporosis and fractures (9-13). In addition, this assessment
is central to health science research and clinical trials.
Physical, emotional, and psychological incapacity, combined

are associated with quality of life after patients undergo
medical or non-medical treatments (27).

with the pain that results from hip, spine, or wrist fractures,
can alter quality of life (14).
After a hip fracture, only 25% of individuals return to
their activities of daily living, such as cooking or going to
the mall (15). The loss of independence that results from the
inability to walk (caused by functional limitations or by the
fear of falling) is the principal consequence of a hip fracture.
This inactivity worsens osteoporosis and increases the risks
of falling and suffering new fractures (16).
The functional alterations caused by a vertebral fracture
can reduce the ability of patients to perform the activities of
daily living at home and care for themselves, which
increases the fear of falling and the risk of new fractures
(17,18). Vertebral fractures are present in approximately one
third of the elderly Brazilian population (19). Vertebral
fractures are strongly associated with lumbar pain and
functional limitations (20,21). In addition, such fractures
reduce lung, heart, stomach, and urinary capacities, thereby
significantly reducing the quality of life (22).
During the period following a wrist fracture, the
individual can experience pain and movement limitations.
Certain activities may be restricted, and such individuals
could have chronic pain and reduced functions (23).
Fracture events can affect the physical and mental
domains of quality of life to different degrees depending
on the type and severity of the fracture (24). Fracture
patients experience psychological sequelae, such as anxiety,
fear, depression, reduced self-esteem, and social isolation
(8).
By measuring quality of life, we can predict clinical
evolution and functional changes, as well as understand the
conditions that will lead to developing better osteoporosis
treatments, thereby improving patient health, reversing
bone loss and reducing the risk of fractures.
Therefore, the search for osteoporosis-targeted questionnaires is essential to monitor and therapeutically evaluate
individuals who suffer from this metabolic bone disease.

Osteoporosis Quality of Life Questionnaire
The Osteoporosis Quality of Life Questionnaire (OQLQ) is
administered during an approximately 20-min interview.
The questionnaire consists of 30 items that are distributed
into five domains: symptoms, physical function, activities of
daily living, emotional function, and leisure. All of the
patients selected for the OQLQ development were diagnosed with chronic lower back pain (28) and osteoporosis
and had previously suffered moderate or severe vertebral
fractures (11). The OQLQ is used to evaluate pharmacological treatments and physical rehabilitation programs.
Studies have reported that this questionnaire correlates well
with generic measures and fractures and is able to detect
health improvements or disabilities (17).

Mini-Osteoporosis Quality of Life Questionnaire
The Mini-Osteoporosis Quality of Life Questionnaire
(mini-OQLQ) was developed to reduce the time needed in
clinical practice to apply the OQLQ (29). The mini-OQLQ is
a 10-item abbreviated form of the original 30-item OQLQ.
Nevertheless, the mini-OQLQ comprises the same five
domains: symptoms, physical function, activities of daily
living, emotional function, and leisure. The mini-OQLQ
is a self-reported questionnaire that requires approximately
3 min to complete.

Osteoporosis Assessment Questionnaire
The Osteoporosis Assessment Questionnaire (OPAQ) is
a self-reported instrument that assesses quality of life in
postmenopausal women with osteoporosis and fractures
(30), and it was recently used by our group to analyze the
quality of life after a balance training program in women with
senile osteoporosis (31,32). The OPAQ comprises five
questions that assess overall well-being and another 79
questions that are grouped into 18 domains. These domains
are distributed into four dimensions (33): physical function,
psychological status, symptoms, and social interaction.
Cantarelli (34) adapted and validated this questionnaire for
use in Brazil (in Brazilian Portuguese) and demonstrated that
the OPAQ is a valid and reproducible instrument to evaluate
osteoporosis patients. Because the OPAQ comprises a large
number of questions and requires 30-40 min to complete,
revised versions of the questionnaire have been developed,
namely the Osteoporosis Assessment Questionnaire 2
(OPAQ2) and the Osteoporosis Assessment Questionnaire
Short Version (OPAQ SV).

MATERIALS AND METHODS
The systematic search for quality of life questionnaires
was conducted in Medline, the Scientific Electronic Library
Online database, and the Latin-American and Caribbean
Health Sciences Literature database; data over the last 20
years (January 1991-Janauary 2011) was searched using the
following MeSH terms: osteoporosis, quality of life, questionnaires, and instruments.

Specific instruments
In recent decades, specific instruments that measure
quality of life in osteoporosis patients have been developed.
Nine questionnaires were initially developed, and another
five were derived from those nine.

Osteoporosis Assessment Questionnaire 2
The OPAQ2 comprises 67 items grouped into 14 health
scales (26). The OPAQ2 is a self-reported questionnaire that
has been tested in elderly populations (35), and it requires
20-30 min to complete.

Womenâ&#x20AC;&#x2122;s Health Questionnaire
The Womenâ&#x20AC;&#x2122;s Health Questionnaire (WHQ) was developed to evaluate menopausal and postmenopausal women
(25). It covers specific symptoms that are observed during
this phase of life. The WHQ is reproducible and exhibits
excellent correlations with estrogen levels and other qualityof-life scales (8,26). The WHQ has been tested for its ability
to assess the efficacy of interventions and has exhibited high
sensitivity for detecting changes in specific symptoms that

Osteoporosis Assessment Questionnaire Short
Version
The OPAQ SV consists of 34 items organized into three
dimensions: physical function, emotional status, and symptoms (36). The OPAQ SV does not collect data related to the
patientâ&#x20AC;&#x2122;s daily activities or social status (37).

adapted to the lifestyle of the Japanese people (43). The
JOQOL comprises 38 items that are grouped into six
domains: pain, activities of daily living, social activity and
leisure, general health, postural awareness, psychological
factors, and falls (44).

Osteoporosis Functional Disability Questionnaire
The Osteoporosis Functional Disability Questionnaire
(OFDQ) (38) was developed to assess functional disabilities
in osteoporosis patients who experienced vertebral compression and lower back pain caused by vertebral fractures. The
OFDQ has been evaluated in clinical trials involving exercise
programs (38) and proved sensitive for detecting improvements in the activities of daily living among the patients in
rehabilitation programs. The OFDQ is a self-reported questionnaire that requires approximately 25 min to complete. The
questionnaire comprises 59 items grouped into five domains:
pain, depression, functional status, social activities, and
confidence in the treatment proposed. The OFDQ has proven
useful in assessing clinical severity and exhibits a reliable
correlation with spinal injury caused by osteoporosis (8,33,38).

16-item Assessment of Health-Related Quality of
Life in Osteoporosis
The 16-item Assessment of Health-Related Quality of Life
in Osteoporosis (ECOS-16) is a short questionnaire that is
rapidly applied and easily administered (45). The ECOS-16
comprises 16 questions, four of which are from the OQLQ,
and 12 of which are from the QUALEFFO. These 16
questions are grouped into four categories: physical function, disease-related fear, psychosocial status, and pain. The
ECOS-16 is a self-reported questionnaire with satisfactory
preliminary psychometric properties. The questionnaire
appears to be a promising tool for use in research and
clinical practice when evaluating postmenopausal women
with osteoporosis with or without vertebral fractures (46).

Quality of Life Questionnaire of the European
Foundation for Osteoporosis
The Quality of Life Questionnaire of the European
Foundation for Osteoporosis (QUALEFFO) originally comprised 48 questions, including six visual analogue scales
(39). The QUALEFFO is specific to patients with vertebral
fractures and comprises five domains: pain, physical
function, social function, general health perception, and
mental function (8,40). The questionnaire has been used in
prevention and treatment protocols, and it has proven to be
reproducible and coherent. After the QUALEFFO had been
validated, two summarized versions of it were developed.

Quality of Life Questionnaire in Osteoporosis
The Quality of Life Questionnaire in Osteoporosis
(QUALIOSTTM) was developed in 2001 (47). The
QUALIOST is a specific instrument that is used in
conjunction with the generic Medical Outcomes Study 36item Short-Form Health Survey because the QUALIOST
includes domains that are not addressed by the latter
instrument (fear of the future, self-image, well-being,
mobility, localized pain, and specific mental repercussions).
The QUALIOST is a self-reported questionnaire comprising
23 questions that are distributed into two dimensions:
physical and emotional. The questionnaire can be used in
therapeutic trials to assess the impact of vertebral fractures
on the quality of life of women with postmenopausal
osteoporosis.

41-item Quality of Life Questionnaire of the
European Foundation for Osteoporosis
The 41-item Quality of Life Questionnaire of the European
Foundation for Osteoporosis (QUALEFFO-41) was developed
to measure the quality of life in patients with vertebral
deformities (41). The QUALEFFO-41 consists of 41 questions
grouped into five domains: pain, physical function, social
function, general health perception, and mental function (8,39).

DISCUSSION

31-item Quality of Life Questionnaire of the
European Foundation for Osteoporosis

Indications of osteoporosis-targeted quality-of-life
questionnaires

The 31-item Quality of Life Questionnaire of the European
Foundation for Osteoporosis (QUALEFFO-31) was developed as a shortened version of the QUALEFFO-41 (41). The
QUALEFFO-31, which consists of three domains (pain,
physical function, and mental state), excludes the most
redundant questions of the QUALEFFO-41 and improves its
conceptual structure.

Perimenopause
The WHQ should be used to evaluate women in
perimenopause because it addresses the specific characteristics of this population. A disadvantage of the WHQ is that
it does not address the feelings that result from social
interactions and is restricted to evaluating how women
perceive the perimenopause-related alterations in their
bodies (48).

Osteoporosis-targeted quality of life
The Osteoporosis-Targeted Quality of Life (OPTQoL)
questionnaire is used in epidemiological studies assessing
the quality of life of elderly women with or without clinical
osteoporosis (39). The OPTQoL questionnaire is a reliable
instrument that comprises 26 scored questions that are
distributed in three domains (physical activity, adaptations
for activities of daily living, and fears) and six additional
questions regarding the clinical and diagnostic alterations of
osteoporosis (5,8,42).

Fractures: comparisons of specific questionnaires
The most extensively tested questionnaires regarding
vertebral fractures are the OQLQ (interviewer-administered) and the QUALEFFO (self-report). The OQLQ has
been tested in patients with osteoporosis and fractures
associated with chronic lower back pain. The QUALEFFO
has been tested in patients with osteoporosis and fractures
with or without chronic lower back pain. These two
questionnaires were developed as instruments to be used
in evaluating the outcomes of clinical trials. The OQLQ and
the QUALEFFO were compared in a study assessing the
quality of life in women with osteoporosis with vertebral

Japanese Osteoporosis Quality of Life
Questionnaire
The Japanese Osteoporosis Quality of Life Questionnaire
(JOQOL) was based on the OPAQ and QUALEFFO-41 and

fractures (49). The authors found that the performance of
the OQLQ was superior to that of the QUALEFFO. This
evaluation was in part attributed to the fact that the
QUALEFFO is a self-reported questionnaire. The studied
population took longer to complete the QUALEFFO, and a
greater number of questions were left unanswered on the
QUALEFFO than on the OQLQ. In addition, it was observed
that the degree of difficulty in completing the QUALEFFO
was inversely proportional to the patient’s level of education. Furthermore, the psychometric properties of the OQLQ
were found to be significantly superior to those of the
QUALEFFO when evaluating women with one or more
vertebral fractures, a result that was also reported by other
authors (49,50).
In its original form, the OPAQ was largely unsuccessful
within the scientific community for clinical practice and
research purposes because it was quite extensive and timeconsuming. Therefore, shorter versions of this questionnaire
were developed: the OPAQ2 (26) and the OPAQ SV (36).
The OPAQ2 was initially used to evaluate hip fracture cases
(35).

2)

3)

Mode of administration: self-reported or
interviewer-administered?
In clinical practice, self-reported questionnaires are an
excellent option because patients can complete such questionnaires in the waiting room. However, this procedure
depends on the patient’s level of education.

4)

Time required for questionnaire administration
The time required to complete a questionnaire is
dependent on the behavior of the patient and the physician.
Short questionnaires can be easily completed by the patient
in a short period of time, thus increasing the patient’s
willingness to do so. The mini-OQLQ, for instance, requires
2-3 min to complete (29). The mini-OQLQ is considered a
sensitive instrument for evaluating patients with osteoporosis, vertebral fractures and pain, (51) as well as postmenopausal women with osteoporosis and vertebral fractures
(52). However, one study demonstrated that the miniOQLQ score exhibits a weak correlation with the clinical
severity of the disease (11). Another short questionnaire is
the ECOS-16. The ECOS-16 comprises 16 questions, has
adequate preliminary psychometric properties and seems
promising for use in research and clinical practice when
evaluating women with postmenopausal osteoporosis with
or without vertebral fractures.

Assessing quality of life is essential to health research and
clinical trials involving osteoporosis. The choice of the
instrument used to assess quality of life depends on the type
of research and on the research question asked; each
instrument has specific advantages and disadvantages (8).
Furthermore, it is important that these instruments be
available in the patient’s native language because a specific
methodology has been established to validate their use
(2,55,56).
Most quality of life osteoporosis questionnaires have been
developed in the English language (33,42,28). Thus, for these
instruments to be used in international studies and in
clinical practice, it is necessary that these instruments
address the same concepts in all languages to make it
possible to pool data and compare results across countries.
In fact, these nine questionnaires should be validated and
proven reliable before being used.
Indeed, many questionnaires have already been validated
for use in other countries and/or cultures (57-61). The
QUALEFFO (39) is the quality of life osteoporosis instrument most validated in other countries, including the
following languages: Serbian (7), Turkish (58), Chinese
(59), Spanish (60), and Italian (61). Only the OPAQ
instrument has been validated in Portuguese (34).
Measuring health-related quality of life has become an
important issue in health service research and in clinical

Focus on patient adaptations
If the
patients
activities
tionnaire

assessment focuses on disability, the need for
to make adaptations to perform daily living
and related patient concerns, the OPTQoL quesshould be used.

Effects of exercise on quality of life using
osteoporosis-specific questionnaires
Only three studies have used specific questionnaires to
assess the effect of exercise on quality of life (35,51,52).
1)

correlated significantly with the disease. By applying
the OFDQ, the authors were able to detect significant
improvements in the performance of daily living
activities and social interactions as well as reduced
pain in patients who performed aerobic exercises.
However, osteoporosis patients who were sedentary
exhibited increased pain and reduced abilities to
perform activities of daily living (37). It would be
interesting to apply the OFDQ in studies assessing the
effect of exercise on disability. However, this effect
was not described by the authors who developed the
OFDQ, making it impossible for the scientific community to use the questionnaire for that purpose.
The second study used the OQLQ to assess the efficacy
of a six-month in-home exercise program (stretching,
strength training and walking) in fragile elderly
women with vertebral fractures (53). The authors
observed an improvement in quality of life in terms
of the symptoms, emotional aspect, leisure and social
activity, as well as a reduction in fatigue and pain
when walking.
The third study demonstrated the reproducibility of
the QUALEFFO (54). By administering this questionnaire, the authors observed that resistance training
and agility training significantly improved the quality
of life, social interaction, physical ability and back pain
of elderly women with osteopenia or osteoporosis.
The fourth study, conducted by our group, demonstrated that over a 12-month period, the Balance
Training Program reduces falls and improves functional balance (31) and quality of life (32). The quality
of life was evaluated before and at the end of the trial
using the Osteoporosis Assessment Questionnaire
(OPAQ) and demonstrated an improvement in the
followings domains: well-being, physical function,
psychological status, symptoms and social interactions
(32).

The first study assessed quality of life after patients
engaged in an exercise program (37). The authors of
the study developed the OFDQ to determine whether
disability and back pain caused by vertebral fractures

trials involving osteoporosis. Nine specific questionnaires
related to osteoporosis (OP) quality of life are available in
the literature. The choice of a particular questionnaire
(WHQ, OQLQ, OPAQ, OFDQ, QUALEFFO, OPTQOL,
JOQOL, ECOS-16, and QUALIOSTTM) will depend on the
type of research and the major question being asked because
each instrument may have particular advantages. It is
important that all of these OP-specific questionnaires be
validated in the language of the country of origin before
being used in clinical research and clinical practice.
Key points:

N
N
N
N
N

Nine specific questionnaires related to osteoporosis (OP)
quality of life are available in the literature.
QUALEFO is the OP-specific questionnaire most commonly used in the literature.
QUALEFFO and OQLQ are targeted more toward
fracture assessments.
OFDQ is used in longitudinal studies involving exercise.
Osteoporosis-specific quality of life questionnaires
should be validated in the language of the country of
origin before being used.

ACKNOWLEDGMENTS
We are grateful to CNPq (300559/2009-7 to RMRP) and CAPES (to
MMM) for providing the funding for this review. Neither CNPq nor
CAPES played any part in the completion of the review or in this paper.

AUTHOR CONTRIBUTIONS
Pereira RM and Madureira MM were responsible for the study concept
and design. Madureira MM and Pereira RM conducted the analysis and
interpretation of the data. Madureira MM, Ciconelli R, and Pereira RM
prepared and revised the manuscript.

antibiotic modifications. This article presents a non-systematic, state-of-the-art review of the biological and clinical
features of CAP biomarkers.

INTRODUCTION
Approximately 4 million adults develop communityacquired pneumonia (CAP) in the United States (U.S.)
annually; CAP is also the eighth leading cause of death in
the U.S. (1). Severe CAP is responsible for 6.6% to 16.7% of
pneumonia hospitalizations in Europe and the U.S. (2,3).
The highest mortality rates, between 20% and 50%, are
observed in severe CAP infections in Spanish and British
intensive care units (ICUs) (4,5).
Hospitalized CAP patients undergo clinical, radiological
and laboratory tests to determine the disease severity,
need for ICU hospitalization and possible complications.
Hemograms, urea, creatinine, glucose, hepatic function tests,
pulse oximetry, arterial blood gasometry and blood and
sputum cultures are critically important (6,7). Identifying the
etiological agent has no relevant effect on the hospitalization
time or mortality in the first 30 days or between the
comparisons of focused therapy and the identified agent or
empirical therapy across a large spectrum (8).
Severity scores, such as the Pneumonia Severity Index (PSI)
and CURB-65 (confusion, urea, respiratory rate, arterial
blood pressure and age) scores, have been developed and
validated. These scores can aid the decision-making process
of hospitalization and ICU referral (9).
Biomarkers are useful tools in the diagnosis, prognostics
and follow-up treatment of CAP and for investigating

Procalcitonin
Procalcitonin (PCT) is a protein that is encoded by the
CALC-I gene on chromosome 11, which produces calcitonin
and several additional free peptides after several posttranslational modifications (10).
PCT concentrations in the serum of healthy subjects are
undetectable or low, generally ,0.1 ng/mL (11). PCT is
detected in other tissues in healthy subjects, but the
transcription of the extra-thyroid CALC-I gene is poor in
the absence of infection. PCT mRNA is up-regulated in
sepsis, which increases the expression and secretion of this
peptide in tissue (10).
Inflammatory and infectious injuries stimulate the
increase in serum PCT (11). The synthesis of this peptide is
particularly induced during severe bacterial infection, sepsis,
septic shock and multiple organ dysfunction syndrome (12).
PCT supports a CAP diagnosis, and this protein is a
predictor of complications and mortality. PCT and C-reactive
protein (CRP) enhance the diagnostic accuracy of the clinical
signs and symptoms that are routinely used for screening
and diagnosing CAP (13). The standard clinical model
exhibited a diagnosis accuracy of 0.79 (IC 95% 0.75–0.83) in
this study, and including these biomarkers increased the
accuracy to 0.92 (IC 95% 0.89–0.94), which was significantly
better than the association of one of these biomarkers alone
(p,0.001 for both comparisons).
Boussekey et al. (11) have also evaluated the prognostic
value of PCT for CAP and demonstrated that PCT .2 ng/mL
was associated with an increased incidence of bacteremia,

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

1321

Biomarkers in CAP: State-of-the-Art Review
Seligman R et al.

CLINICS 2012;67(11):1321-1325

associated with resistant strains and reducing hospitalization costs.

septic shock, multi-organ failure and mortality. No association for CRP was observed. Antibiotic administration must be
based on the PCT cutoff ranges (14). Antibiotic treatment is
intensified when the infection is severe and the PCT levels
remain elevated (.0.25 or 0.5 ng/L). Antibiotics may be
discontinued when the PCT levels decrease rapidly.
Christ-Crain et al. (15) demonstrated that using PCT for
therapeutic guidance substantially reduced total antibiotic
exposure and decreased the treatment duration by 55%
compared to the standard therapeutic treatment (median 12
days vs. 5 days, p,0.001). Reduced adverse effects and
microbiological resistance rates and shortened antibiotic
therapy courses improve resource allocations, which is an
important factor in public healthcare.

Copeptin
Arginine-vasopressin (AVP) is a hormone that is produced in the paraventricular nuclei of the hypothalamus
and stored in the posterior part of the pituitary gland.
Several stimuli, such as hypotension, hypoxia, hyperosmolarity, acidosis and infections, stimulate the release of AVP
(23). AVP is released into the circulatory system by osmotic
and hemodynamic stimuli. AVP exerts antidiuretic and
vasopressor effects, which may restore the vascular tonus in
vasodilatation hypotension (24).
Copeptin is a 39-amino acid glycopeptide, and its
physiological function is unknown. AVP and neurophysin
II comprise the terminal portion of the pre-pro-vasopressin
molecule (25). Copeptin may play an important role in the
correct structural formation of the AVP precursor, which is
required for its proteolytic maturation efficiency (26).
Serum AVP levels have limitations because of the short
half-life of AVP and its molecular instability. However,
copeptin is highly stable ex vivo even for several days at
room temperature. Ex vivo copeptin may be an indirect
parameter to estimate the AVP plasma concentrations in
critical patients, including the patients with sepsis and
septic shock, for whom the levels of these biomarkers are
high (27,28).
The presence of copeptin indicates the need for follow-up
treatment for different types of pneumonia. Copeptin may
be an independent predictor of mortality in CAP. CAP was
an independent predictor of mortality in ventilationassociated pneumonia, and mortality rates increased with
the severity of the sepsis (29).

C-Reactive Protein
C-reactive protein (CRP) was the first ‘‘acute phase’’
protein to be described (16). CRP was discovered in the
serum of patients with pneumococcal pneumonia; the CRP
precipitated at the C-polysaccharides from the bacterial
membrane. Combining CRP with the phosphocholine
molecule responded to C-polysaccharide and other bacterial
and host cell membrane constituents. Other ligands have
also been described.
CRP activates the classical complement pathway, stimulates phagocytosis, binds to the immunoglobulin receptors,
and interacts with several molecules (17). CRP values
,3 mg/L are normal, and values.10 mg/L indicate significant inflammation (18). CRP is a sensitive inflammatory
biomarker, but it exhibits low specificity. CRP values
between 3 mg/L and 10 mg/L may reflect numerous
conditions, such as obesity, smoking, diabetes mellitus,
uremia, hypertension, low physical activity, oral hormone
replacement therapy, sleep disturbances, chronic fatigue,
alcohol consumption, depression, aging and other states that
do not necessarily include inflammation (19).
A cut-off point of 11 mg/L serum CPR demonstrated a
94% sensitivity and 95% specificity in healthy individuals
and CRP patients, respectively. These data suggest that CPR
values below this point may exclude a confirmed CAP
diagnosis. With an 83% sensitivity and 44% specificity, a
cut-off point of 33 mg/L CRP distinguished the patients
with a confirmed CAP diagnosis from the patients with
similar clinical symptoms but different clinical conditions
(20).
Chalmers et al. (21) concluded that CRP values ,100 mg/
L in CAP patients on the day of admission and four days
later were independently associated with a low 30-day
mortality rate, low probability for mechanical ventilation
and/or inotropic support and low rates of complicated
pneumonia. The risks of 30-day mortality, need for mechanical ventilation and/or inotropic support and complicated
pneumonia increased when the CPR levels did not drop by at
least 50% until the fourth day of admission.
A cohort of 53 subjects (22) demonstrated that daily
measurements of serum CRP in the patients with severe
CAP are useful for identifying the patients with a poor
prognosis, and this biomarker is a better predictor than the
commonly used markers of infection, such as body
temperature and leukocyte count. This study also demonstrated that shorter antibiotic therapy might exhibit the
same efficacy with less toxicity in patients with a rapid drop
in CRP levels, thereby avoiding the emergencies that are

Pro-ANP
Members of the family of natriuretic peptides are
established biomarkers for congestive heart failure (30).
These proteins defend the body against hypertension and
salt and water retention by antagonizing the renin-angiotensin-aldosterone system. Natriuretic proteins alter renal
sodium reabsorption, vascular tonus and cell growth. The
smooth muscles of the blood vessels and kidneys are the
primary targets of atrial natriuretic peptide (ANP). ANP
distends the smooth muscles of the vessels, and increases
the permeability of capillaries, which facilitates the removal
of water and sodium. This hormone also inhibits the
function of several other hormones, such as endothelin
and vasopressin (31).
ANP is predominantly produced in the atrium of the heart,
and this peptide comprises 98% of the natriuretic peptides in
circulation. The pre-pro-ANP hormone is composed of 151
amino acids. The amino acid chain is called pro-ANP after
removing a 25-amino acids signal sequence. The pro-ANP is
likely cleaved by the membrane proteins in a functional ANP
chain to a 28-amino acid peptide and an amino-terminal
fragment of 98 amino acids (the NT-pro-ANP) prior to
exocytosis (32).
Distended atrial walls signal the ANP release. High
cardiac output, sympathetic stimulation and metabolic
factors influence the ANP release. It is also suspected that
hypoxia influences the ANP release. The half-life of ANP is 2
to 5 minutes, and its degradation rate is approximately 14 to
25 mL/min/kg (33).

1322

CLINICS 2012;67(11):1321-1325

Biomarkers in CAP: State-of-the-Art Review
Seligman R et al.

ANP is a marker for the prevention and differential
diagnosis of several diseases. The use of this peptide in
diagnosing dyspnea caused by heart failure is more efficient
than traditional methods (34). ANP and pro-ANP are
interesting new sepsis and pneumonia markers (35,36).
Morgenthaler et al. (37) have compared the pro-ANP levels
to the APACHE II score (Acute Physiology and Chronic
Health Evaluation) as an outcome predictor in septic
patients.

Cortisol
The hypothalamic-pituitary-adrenal circuit is activated by
central stress control circuits to produce and secrete the
corticotropin-releasing hormone (CRH). CRH stimulates the
anterior portion of the pituitary gland to synthesize and
release proopiomelanocortin (POMC), an adrenocorticotropin (ACTH) precursor. In the systemic circulation, ACTH
activates the transcription of steroids, particularly cortisol,
in the adrenal gland (47).
Cortisol secretion increases in amplitude but not frequency after three to five hours of sleep, and secretion peaks
a few hours before waking until one hour after waking.
Cortisol amplitude decreases in the morning and reaches a
minimum level at dawn (48). The half-life of cortisol is
approximately 80 minutes, which is longer than the 8minute half-life of ACTH (49). The plasma cortisol levels are
higher in cases of severe trauma, burns, major surgery,
hypoglycemia, fever, blood pressure changes, exercise and
exposure to intense cold (50-53).
Salluh et al. have demonstrated that treatment with
supraphysiological doses of hydrocortisone increases the
survival rates of severe CAP patients who develop adrenal
failure during septic shock (54). A study of 72 CAP patients
demonstrated that the baseline level of total cortisol was
significantly higher in non-survivors. These results confirm
the interference of infection in adrenal functions and
support the value of cortisol as a better predictor of
mortality compared to severity-related scores (APACHE II,
CURB-65, SOFA) and laboratory markers (CRP, leukocyte
count, and d-dimers) (55).

Adrenomedullin
Human adrenomedullin (ADM) is a 52-amino acid
peptide that is synthesized as part of pre-pro-adrenomedullin, a larger precursor molecule (38). The ADM gene is
expressed in a wide range of tissues, but initial studies on
the distribution of this gene have suggested that the highest
levels of expression are observed in the adrenal medulla,
ventricular chambers, kidneys and lungs (39). The ADM
gene is more highly expressed in the endothelial cells than
the adrenal medulla, and this peptide is a secretory product
of the vascular endothelium, which also includes nitric
oxide (NO) and endothelin (40).
The plasma half-life of ADM is approximately 22 minutes
(41). The normal plasma concentrations of ADM range from
1 to 10 ng/mL, and most values are between 2 and 3.5 ng/
mL (42). However, obtaining reliable measurements of
ADM release in blood circulation is difficult because ADM
immediately binds to receptors near the site of its production. The short half-life of ADM and technical difficulties
also complicate the plasma measurements (43).
The plasma ADM levels are elevated in a wide range of
disease states, usually as a compensatory response to
cardiovascular disturbances (42). ADM likely participates
in the physiopathology of septic shock because this is the
only pathological condition in which the plasma levels of
this protein approach the levels that are required for
receptor activation. The ADM plasma levels in sepsis
patients are directly responsible for hypotension during
septic shock (44).
Christ-Crain et al. (45) have noted that the levels of MRpro-ADM on admission increased according to the CAP
severity (based on the PSI score). MR-pro-ADM is a stable,
functionally irrelevant fragment of ADM degradation that is
used in some studies because of its better technical viability.
This progressive increase was also observed in procalcitonin
(p,0.0001). However, no statistical significance was
observed for the C-reactive protein, total leukocyte count,
and body temperature.
The ADM levels upon admission were significantly
higher in the patients who died during the follow-up
compared to the patients who survived: 2.1 (1.5–3.0 nmol/
L) vs. 1.0 (0.6–1.6 nmol/L) (p,0.001). An analysis of the
‘‘treatment failure’’ and ‘‘death’’ outcomes demonstrated
that the prognostic accuracy of ADM was similar to the PSI
score but higher than other parameters (44).
Kru¨ger et al. have demonstrated that the MR-proANP
(mid-regional pro-atrial natriuretic peptide), copeptin, CTproET-1 (proendothelin-1), and MR-proADM (mid-regional
proadrenomedullin) biomarkers are strong predictors of the
28- and 180-day CAP mortality, and MR-proADM exhibited
the best performance. The combination of CRB-65 and MRproADM was the best predictor for short- and long-term
mortality (46).

D-dimers
D-dimers are released into the blood during the dissolution process of fibrin emboli in the fibrinolytic system. Ddimers are the smallest fragments of the fibrin degradation,
and these proteins are detectable in blood plasma. The halflife of this protein is approximately 8 hours, and it is cleared
from the plasma via urinary excretion and the action of the
reticuloendothelial system (56).
High d-dimers levels have been detected in patients with
disseminated intravascular coagulation (DIC), severe sepsis,
thrombotic events, hepatic diseases, surgery and trauma
(57-59). The most important application of d-dimers is
related to thrombotic events. D-dimers have been studied
extensively as a diagnostic method for deep vein thrombosis
(DVT) and pulmonary embolism (PE). A negative result has
diagnostic utility that is comparable to normal lung scans or
negative duplex ultrasound findings (60).
The application of the d-dimers analysis to CAP is a novel
approach. In a cohort study of 68 CAP patients, Shilon et al.
have demonstrated a positive correlation between d-dimers
and PSI, APACHE II, hospitalization time, organ failure,
fever duration, and hospital mortality (61). Another study of
302 CAP patients (62) investigated the relationships
between plasma d-dimers levels and the prognostic variables that are included in the PSI. High d-dimers levels were
associated with radiological pneumonia extension findings.
Using biomarkers may aid in the diagnosis, treatment
and prognosis of CAP. Table 1 summarizes the reviewed
biomarkers and triggers. The PCT serum levels may provide
valuable support to the clinical diagnosis of CAP and aid in
the differential diagnosis of bacterial and viral pneumonia.
PCT is particularly useful because the results are obtained
several days prior to the culture tests. These biomarkers also

aid in identifying the low-risk patients who can be treated in
outpatient environments. Reducing unnecessary hospitalizations decreases treatment costs and patient discomfort.
Protocols based on PCT levels can substantially reduce
the use of antibiotics and treatment times. Antibiotic
prescriptions can be encouraged or discouraged via the
use of PCT serum levels. The clinical course of pneumonia is
reflected in the serum levels of PCT and CRP. CRP is
already a widely used biomarker during the follow-up of
infectious processes, and it is included in the clinical
protocols of several hospitals. Decreasing the levels of these
biomarkers is critical to predicting patient survival, and
increased biomarker levels indicate the progression to septic
shock, multiple organ failure and death. New biomarkers,
such as pro-ANP and copeptin, are under investigation, and
these markers demonstrate effective prognostic powers.
Finally, PCT is currently the most appropriate biomarker.
PCT distinguishes cases according to their severity, and the
PCT levels may direct the treatment of complicated cases.
PCT levels rise in proportion to the severity of the bacterial
infection, but the levels do not increase in viral infections.
Therefore, low PCT levels preclude the need for antibiotics.
Elevated PCT levels are associated with an increased rate of
bacteremia, septic shock, multi-organ failure and mortality.
Decreasing PCT levels during antimicrobial treatment
indicate a favorable outcome with a lowered risk of death.
The behavior of infections remains unclear. Biomarkers
may assist clinicians in determining the severity of the
patient symptoms in these diseases.

ACKNOWLEDGMENTS
We would like to thank the Post-Graduation and Research Group (Grupo de
Pesquisa e Po´s-Graduac¸a˜o-GPPG) of Hospital de Clı´nicas de Porto Alegre.

AUTHOR CONTRIBUTIONS
All of the authors were equally involved in the bibliographic revision, data
compilation and manuscript writing and revision.

Although the diagnosis of Graves’ orbitopathy is primarily made clinically based on laboratory tests indicative of
thyroid dysfunction and autoimmunity, imaging studies, such as computed tomography, magnetic resonance
imaging, ultrasound and color Doppler imaging, play an important role both in the diagnosis and follow-up after
clinical or surgical treatment of the disease. Imaging studies can be used to evaluate morphological abnormalities of
the orbital structures during the diagnostic workup when a differential diagnosis versus other orbital diseases is
needed. Imaging may also be useful to distinguish the inflammatory early stage from the inactive stage of the disease.
Finally, imaging studies can be of great help in identifying patients prone to develop dysthyroid optic neuropathy and
therefore enabling the timely diagnosis and treatment of the condition, avoiding permanent visual loss. In this paper,
we review the imaging modalities that aid in the diagnosis and management of Graves’ orbitopathy, with special
emphasis on the diagnosis of optic nerve dysfunction in this condition.
KEYWORDS: Graves’ Ophthalmopathy; Optic Nerve Diseases; Multidetector Computed Tomography; Magnetic
Resonance Imaging; Ultrasonography; Color Doppler Ultrasonography.
Gonc¸alves ACP, Gebrim EM, Monteiro ML. Imaging studies for diagnosing Graves’ orbitopathy and dysthyroid optic neuropathy. Clinics.
2012;67(11):1327-1334.
Received for publication on July 13, 2012; First review completed on July 13, 2012; Accepted for publication on July 30, 2012
E-mail: allanpieroni@uol.com.br
Tel.: 55 11 3081-2199

pose few diagnostic difficulties when these characteristic
ocular findings occur concomitantly with the thyroid disease.
However, when unilateral or inconclusive ocular features
occur in the absence of objective evidence of thyroid
dysfunction, GO can be difficult to diagnose (6).
Among the ocular features, eyelid retraction plays a major
role in the clinical diagnosis of the disease. According to
Bartley and Gorman’s diagnostic criteria (7), GO may be
diagnosed when eyelid retraction occurs in association with
exophthalmos, DON or extraocular muscle involvement. If
eyelid retraction is absent, positive laboratory tests are
required for diagnosis.
Affecting 4–8% of patients, DON has long been recognized
as the most feared complication of GO (8,9). Although
inflammatory (10–12) and ischemic (13) mechanisms have
been suggested, the most widely accepted explanation is the
mechanical compression of the optic nerve at the orbital apex
by the enlarged extraocular muscles (14). Because DON may
present a wide range of symptoms and signs, its diagnosis
depends on several clinical features, including decreased
visual acuity (VA), abnormal visual fields (VF), impaired
color and brightness perception, delayed visually evoked
potentials, afferent pupillary defects and edema or atrophy of
the optic nerve head (9). Patients with GO are often assumed
to have DON when one of these features is present and no
other cause for the defect is observed, but visual impairment
in GO is not uncommonly related with other factors (15).
Consequently, in GO patients, direct optic nerve function
testing can yield misleading results that occasionally make it
difficult to distinguish probable from definitive DON.

INTRODUCTION
Graves’ orbitopathy (GO) is the most common extrathyroidal manifestation of Graves’ disease (GD), occurring in
25–50% of patients with the disease (1,2). GO may occur
during or after the onset of hyperthyroidism and less
frequently, in euthyroid or hypothyroid patients. The close
clinical association between immunogenic hyperthyroidism
and orbitopathy suggests that the antigen responsible for
these diverse conditions may be shared by the thyroid gland
and orbital tissues (3). The disease has a self-limited active
phase that usually lasts 18 to 24 months and abates slowly,
followed by an inactive (static) phase (4). In the active phase,
inflammation, the accumulation of glycosaminoglycans and
an increased fat content determine the tissue expansion
within the relatively fixed space constraint of the bony orbit.
The diagnosis of GO is usually made clinically. The signs
and symptoms of active GO include lid retraction, proptosis,
conjunctival injection, chemosis, diplopia, corneal ulceration
and rarely, dysthyroid optic neuropathy (DON) (5). In the
chronic fibrotic phase, lid retraction, proptosis and restrictive
strabismus are the most common findings. Patients with GO

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

to their original polarity, emitting a measurable amount of
energy. This process is called T1 relaxation (T1), and it is best
measured just after the radio-frequency excitation is stopped.
Radio-frequency excitation also initiates a uniform synchronous precession, or spin, in the protons. Following the
excitation, the spinning subsides at different rates in different
molecular environments. The energy emission measured
from this process is called the T2 relaxation time (T2) (4). T1
and T2 can be used to distinguish between tissue types with
different proton densities. T1-weighted (T1w) images may be
used to evaluate anatomic structures, whereas T2-weighted
(T2w) images provide useful information about tissue composition. Additionally, certain T1 or T2 weights achieved by
applying special pre-pulses can be used to distinguish water
from fat.
Ultrasonography (US). Grayscale US has been used in
ophthalmology since the late 1950s. Standardized diagnostic
US for eye diseases is performed using high frequencies
(optimally 8 MHz) and small wavelengths to visualize small
ocular structures. Both A-scan and B-scan transocular
echograms are performed. A-scans are used to assess the
tissue characteristics based on the reflected acoustic waves.
This technique is particularly sensitive for identifying the
thickening or thinning of the muscles and for differentiating
underlying pathologies. The reflectivity of the extraocular
muscles may change as a result of tissue edema and cellular
infiltration (4). It is easier to visualize the orbital structures
using a B-scan, especially when the examination is not
performed by an experienced ultrasonographer. B-scans are
very helpful in topographic evaluations and when
determining whether individual recti muscles are enlarged.
The main advantages of orbital US are its low cost and the
lack of ionizing radiation. Additionally, in experienced
hands, a relatively short examination time is adequate to
monitor the anterior and midorbital therapeutic response.
The main disadvantages of US are the high intra- and
interobserver variability, the inability to adequately visualize the orbital apex and the poor quality of the anatomic
information obtained of the bony orbital walls compared
with the information provided by CT and MRI (18).
Color Doppler imaging (CDI). CDI is an ultrasonic
imaging modality that allows the assessment of blood flow
in real time on a grayscale B-mode background. The technique
was first described in 1979 (22) and is well tolerated and
widely used as a noninvasive imaging technique in many
medical specialties. More recently, CDI has been introduced as
an adjunct to the clinical examination and cross-sectional
imaging for evaluating several pathological conditions in the
orbit. Although the topography of the orbital structures can be
evaluated with grayscale US, CDI makes it possible to assess
the blood flow in the orbital vessels and detect changes in the
perfusion of the orbital arteries and veins (23).
CDI produces conventional grayscale US images together
with information about the direction and velocity of the
blood flow. The velocity data are superimposed onto the
grayscale image by assigning a color scale to the data (24).
The indications for and uses of CDI in ophthalmology are still
evolving but primarily include vascular disorders. CDI has
been used to investigate changes in blood flow parameters in
disorders such as anterior ischemic optic neuropathy, central
artery occlusion, central retinal vein occlusion, glaucoma,
diabetes mellitus, ocular ischemic syndrome, uveitis and
endophthalmitis. In orbital abnormalities, CDI is well suited
for the evaluation of cavernous-carotid fistula, orbital varix,

Although the diagnosis of GO and DON is based primarily
on clinical signs from laboratory test results suggestive of
thyroid dysfunction and autoimmunity, imaging studies,
such as computed tomography (CT), magnetic resonance
imaging (MRI), ultrasonography (US) and color Doppler
imaging (CDI), can also be extremely important in both the
diagnosis and clinical or surgical follow-up. Imaging studies
can verify possible extraocular muscle involvement as part of
the diagnostic workup and may help distinguish the early
acute inflammatory stage from the fibrotic, inactive stage of
the disease (16). Finally, imaging studies of patients prone to
develop DON make the timely diagnosis and treatment of the
condition possible, avoiding permanent visual loss (17). The
purpose of this paper is therefore to review imaging
modalities that can aid in the diagnosis and management of
GO, with a special emphasis on the diagnosis of GO-related
optic nerve dysfunction.

Imaging modalities in Gravesâ&#x20AC;&#x2122; orbitopathy
Computed tomography (CT). CT can distinguish normal
structures from abnormal structures of different tissue density
based on their differing X-ray absorption properties. Fat and
water have low densities and therefore appear black on CT
images, in contrast to denser muscles, the optic nerve and
bony structures. Effectively acting as a natural contrast
medium, the presence of orbital fat allows good spatial and
density resolution of orbital structures (18). The tissue differences inherent in the orbit obviate the need for intravenous
contrast in many situations. After digital recording, the data
are converted via an arithmetic procedure into different
grayscales. Compared with isodense tissues (e.g., the brain),
tissues with high absorption values (e.g., bone) appear
hyperdense, whereas tissues with low absorption values
(e.g., water or fat) appear hypodense (4).
The introduction of spiral CT in the early 1990s represented a fundamental evolutionary step in the development
and ongoing refinement of CT imaging techniques. Continuous scanning of anatomical regions within a short time
frame yields compelling results, and the technology has
been shown to offer undisputable advantages in lesion
detection and isotropic spatial resolution (19). Individual
volume elements obtained from axial slices can be reformatted in any plane to produce coronal, sagittal, paraxial or
parasagittal oblique images. Unlike direct coronal scans,
sagittal and coronal reformations avoid high spatial
frequency artifacts from dental appliances and other metal
implants. Multiplanar reformations make it possible to view
a lesion in the optimal anatomic plane and determine its
location relative to contiguous orbital, bone, sinus and
central nervous system structures. The advent of multidetector computed tomography (MDCT) has improved
image quality and resolution by enabling the simultaneous
acquisition of multiple slices and faster gantry rotation (20).
Magnetic resonance imaging (MRI). Hydrogen nuclei
with an odd number of nucleons (protons and neutrons)
behave as small magnets or dipoles. Protons are ubiquitous,
and their resonance is the basis of MRI techniques (21). MRI
captures signals from the free-moving protons in tissue as
the protons return to their primary position in a high
magnetic field after deflection by a frequency pulse. When
an organ is placed in a magnetic field, there is a net
alignment of protons. Radio-frequency excitation reverses
the polarity of some of these hydrogen nuclei, raising their
level of energy. When the excitation stops, the protons return

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Imaging studies in Graves’ orbitopathy
Gonc¸alves ACP et al.

orbital tumors, orbital cellulitis and orbital inflammatory
conditions (24,25).
The major blood supply to the orbit is through the
ophthalmic artery (OA), and the major venous drainage is
through the superior ophthalmic vein (SOV) to the cavernous
sinus. CDI can be used to assess the OA and its branches,
such as the SOV and inferior ophthalmic vein. The CDI
assessment of patients with GO is of great value because
studies have shown that venous congestion plays a significant role in the pathogenesis of the disease (26).

enlargement in all four major muscle groups (32). In another
CT scan study involving 349 thyroid patients, the inferior,
medial, superior and lateral recti were enlarged in 43%,
38%, 29%, and 16% of the cases, respectively. Two or more
muscles were enlarged in 70% of the patients with ocular
involvement (37).
Unlike in patients with orbital myositis or idiopathic
orbital inflammation, the evidence of muscle involvement in
patients with GO is usually limited to the nontendinous
portion of the muscle. Additionally, the extraocular muscles
in GO appear to be enlarged in a fusiform fashion, with sharp
borders (38). However, atypical cases with tendon involvement and blurred muscle margins have been described
(31,39–41).
In the evaluation of the extraocular muscle characteristics
in GO, CT, and MRI are the preferred imaging procedures,
although US may also be useful (42). In clinical practice, US
may conveniently be used to measure the extraocular
muscles and exclude other diseases. However, although
some authors have used US to evaluate muscle size, the
technique has been found to have limited accuracy and add
no new information to the knowledge obtained from CT and
MRI studies (37,43,44).
Although the extraocular muscles have been described as
the ‘‘shock organ’’ of GO (31), many studies suggest that
expansion of the orbital fat compartment also represents a
major component of the disease process (45,46). In GO,
although some affected orbits are characterized by prominent
extraocular muscle enlargement, other orbits display mild
or no extraocular muscle involvement, occasionally with
clearly increased adipose tissue volume. Imaging studies can
distinguish these clinical differences (Figure 2). Consequently,
some reported diagnostic criteria for GO include the observation of orbital fat augmentation in CT images (28,47). The
observation of exophthalmos in patients with abnormally

Imaging studies for diagnosing GO and defining
disease activity
Imaging studies can be helpful in establishing the diagnosis of GO because they provide objective morphological
findings of the orbital structures. Based on such studies
(especially MRI and CT), it is possible to establish the degree
of extraocular muscle and orbital fat enlargement, exclude
coexisting orbital pathology, clarify a confusing clinical
picture, and perform surgical planning (12). A CT scan with
positive findings is included in many sets of diagnostic
criteria for GO (7,27–30).
GO presents an unusual imaging pattern. The extraocular
muscles appear to be the primary area of orbital involvement.
Despite attempts to establish normative measurements (31–
34), the assessment of muscle enlargement is often subjective
and requires comparison with the opposite orbit or prior
qualitative experience. Patients with GO usually present
symmetrical, multiple extraocular muscle enlargement in
both orbits, although asymmetrical muscle involvement can
occur. However, true unilateral orbital involvement is
uncommon, occurring in only 6 to 10% of patients (35). The
muscles most frequently affected are the medial and inferior
recti (Figure 1).
In a series of 116 CT scans of a heterogeneous population
of patients with GO in different stages of the disease, 85% of
the patients displayed definitive enlargement of the
extraocular muscles (36). The inferior and medial recti were
involved in 77% and 75% of the cases, respectively, and
were the most severely enlarged. The lateral (51%) and
superior (50%) recti were involved less frequently and less
severely. However, a later study found similar levels of

Figure 2 - Axial CT scans from two patients with Graves’
orbitopathy. A) Patient with prominent enlargement of the
medial and lateral recti muscles. B) Patient with severe proptosis
of both orbits with clear fat tissue augmentation and no
extraocular muscle involvement.

Figure 1 - Coronal CT scans from two patients with Graves’
orbitopathy. A) Patient with symmetric enlargement of the
extraocular muscles in both orbits. B) Patient with asymmetric
involvement of the extraocular muscles.

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Gonc¸alves ACP et al.

CLINICS 2012;67(11):1327-1334

These fatty or fibrotic muscle changes display no contrast
enhancement on matched fat-saturated T1w images.
In the management of GO, it is of great importance to
estimate the disease activity when selecting those patients
most likely to respond to immunosuppressive treatment.
Clinical activity scales, such as the clinical activity score
(CAS) described and validated by Mourits and the
Amsterdam orbitopathy group (57) and VISA classification
described by Dolman and Rootman (58), can be very helpful
in assessing disease activity. Although it might be assumed
that the combination of MRI studies and clinical scores would
improve diagnostic accuracy, the results have been conflicting. Some researchers have found no clear correlation
between MRI findings and CAS indexes (54,59,60), perhaps
because of the great variability in CAS scores between
observers or because only the extraocular muscles, and not
the inflamed orbital fat, were assessed. However, other
researchers have reported significant positive correlations
(61). Despite the controversy, MRI appears to be useful for
monitoring the response to treatment using measurements
such as the signal intensity (SI) and signal intensity ratio
(SIR).
The use of US has also been proposed for evaluating
disease activity. Prummel et al. (62) demonstrated extraocular muscle reflectivity changes in the inflammatory
phase of GO, suggesting that US is a reliable tool for the
determination of disease activity. In the active phase, the
extraocular muscles have a lower internal reflectivity,
presumably due to edema, whereas in end-stage disease,
the muscles tend to show irregular high reflectivity from the
echogenic fibrotic scar tissue. However, not all studies have
found a correlation between CAS and US reflectivity (59,63).
Moreover, US is believed to provide less comprehensive
information on the extraocular muscles and inflammation
than MRI. It should also be noted that the adequate

increased orbital adipose tissue is suggestive of GO, but
obesity and Cushing’s disease should also be considered (48).
GO is associated with a wide spectrum of radiological
findings in addition to extraocular muscle and fat tissue
enlargement, as described in the literature (36,49,50). CT
findings may include bone changes, especially in the lamina
papyracea of the ethmoid, with bowing resulting from
muscle pressure. Lacrimal gland displacement and enlargement, exophthalmos, anterior soft tissue swelling and superior optic vein dilatation may also be observed in imaging
studies, but these are unspecific findings that do not support
the diagnosis of GO (32,51,52).
CT is generally the preferred imaging modality for the
diagnosis of patients with GO because of its ability to
visualize bone and soft tissues in the orbit. CT also aids the
evaluation of the orbital walls, sinus and orbital elements in
orbital decompression planning. Compared with MRI, CT is
less expensive and faster to perform; however, CT is less
efficient in the evaluation of soft tissue changes. Additionally,
MRI can reveal details that may be important in the
assessment of disease activity.
In addition to their importance in the diagnosis of GO,
imaging studies can aid the evaluation of inflammatory
disease activity. Changes observed with CT in sequential
measurements of the extraocular muscles may be related to
clinical activity; muscular involvement occurs early in GO
and subsides together with other clinical signs (53).
Nevertheless, MRI is preferred for studies assessing disease
activity because of its better performance in the evaluation
in soft tissues.
MRI estimates disease activity based on the water content
of the tissues. In GO, strongly T2-weighted and fat-suppressed images obtained using the turbo inversion recovery
magnitude (TIRM) and short tau inversion recovery (STIR)
sequences have been shown to be useful in detecting
extraocular muscle edema (54). To differentiate active from
inactive GO, inflammatory edema of the extraocular muscles
must be distinguished from fibrous end-stage disease with
fatty degeneration using the T2 relaxation time, which is
shorter for fibrous tissue than for inflammatory tissue
(Figure 3) (55). However, edema is not always correlated
with disease activity because edema can also be the result of
both active inflammation and reduced venous outflow in
patients with fibrotic disease. Some studies have shown that
in T1w images, when gadolinium is combined with fat
saturation techniques, it is possible to distinguish inflammatory edema from congestive venous outflow in burned-out
disease (Figure 4) (55,56). Non-fat-saturated T1w images are
also useful in the detection of fatty muscle degeneration.

Figure 3 - Coronal STIR MR image showing a bright signal from
the superior oblique muscles and inferior, medial and superior
recti muscles in both orbits in a patient with active Graves’
orbitopathy.

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Imaging studies in Graves’ orbitopathy
Gonc¸alves ACP et al.

measurement of muscle reflectivity requires standardized
A-wave US equipment and an experienced examiner.
CDI has also been used as a tool for diagnosing GO and
assessing disease activity. Several studies have compared the
orbital blood flow in GO patients and control subjects
(25,64,65), as well as in patients with different clinical forms
of GO (66). Benning et al. (64) found that the flow velocity in
the right ophthalmic artery was much greater in subjects with
clinically active GO than control subjects. This finding was
also reported by Alp et al. (25), which supports the assumption that orbital inflammation increases orbital blood
flow. The research in this area is promising; Monteiro et al.
(26) recently compared the CDI findings from orbits with the
active form of the disease with findings from the same orbits
after (primarily surgical) treatment and found a significant
difference.
Finally, patients with GO may be diagnosed using
octreotide scintigraphy (octreoscan). Octreotide is a somatostatine (SM) analogue labeled with indium, a substance
that has been used to localize tumors with membrane
receptors for SM. Based on the assumption that orbital
lymphocytes express SM receptors during the active phase
of GO, a high uptake of the radiolabelled octreotide may be
correlated with orbital inflammation and active disease
(67,68). A positive orbital octreoscan could be useful in the
assessment of disease activity in GO; however, the fact that
the technique is expensive, non-specific and associated with
a non-negligible radiation burden restricts its clinical
application (4).

Figure 5 - Schematic representation of the method for calculating Barrett’s muscle index. The vertical index was calculated by
the sum of the vertical muscle diameters (A and B) divided by the
height of the orbit (C). The horizontal index was calculated by
dividing the sum of the horizontal muscle diameters (D and E) by
the horizontal diameter of the orbit (F).

In a recent study using MDCT imaging, this linear index
displayed the best combination of sensitivity and specificity
(79% and 72%, respectively) at a muscle index of 60% (71).
Barrett’s index was found by other authors to provide
satisfactory performance in the detection of DON (17,73).
The subjective assessment of apical crowding based on
single coronal images, as described by Nugent et al.(32) and
others (9,17,70,73,74), has also been shown to a be good
predictor of DON. In these studies, a coronal image at the
apex is subjectively graded according to the severity of the
muscle crowding. According to this method, the effacement
of the perineural fat is graded 0 (none), 1 (up to 25%), 2 (25–
50%), or 3 (greater than 50%) (Figure 6) (32). Nugent et al.
found severe apical orbital crowding (grade 3) in 12 of 18
orbits with DON but in only 16 of 124 orbits without DON
(32). Neigel et al. graded 79.2% of the orbits with DON and
12.9% of the orbits without DON as having moderate or
severe crowding (9). Birchall et al. found severe apical
crowding to be a good predictor of DON, with a sensitivity
of 62% and specificity of 91% (74). A recent multicenter
study found apical muscle crowding in 49 of 56 orbits with
DON (70). In another study, severe optic nerve crowding
was retrospectively noted in 80% of the orbits (16 of 20) with
optic neuropathy and 29% of the orbits without DON (10 of
34). No orbits with optic neuropathy had less than grade 2
evidence of optic nerve crowding (17). However, although it
appears clear that the Nugent’s apical crowding score is
useful for raising suspicion of DON, the score does not
provide a clear definition of the position along the orbit
where the coronal plan should be taken to determine the
score, and there is no clear differentiation between grade 1,

Imaging studies for the diagnosis of dysthyroid
optic neuropathy
Computed tomography (CT). Several studies have shown
that certain CT scan parameters increase the suspicion of
DON. Although the stretching of the optic nerve by the
increased orbital fat associated with axial proptosis is in rare
cases considered a possible pathogenic mechanism for
developing optic neuropathy (69), the most important mechanism is orbital apical crowding by the enlarged
extraocular muscles (9,14,17,32,52,70–73). Because the presence of apical crowding in CT images is strongly correlated with
DON in GO, many CT studies have proposed indexes
designed to objectively detect DON in several different
manners.
In a pioneer study based on linear measurements of the
extraocular muscles and bony orbit, Barrett (52) described a
simple method of quantifying extraocular muscle impingement on the optic nerve space. Using a reformatted scan
halfway between the posterior globe and orbital apex, the
vertical diameter of the superior rectus muscle–levator
muscle complex (A) and inferior (B) rectus muscles, as well
as the orbital height (C), were measured with a ruler along a
horizontal line through the optic nerve. The vertical
muscular index was expressed as the percentage of the
orbital height occupied by the superior rectus muscle–
levator muscle complex and inferior rectus muscles ([A+B/
C]6100) (Figure 5). In the same manner, the transverse
dimensions of the medial and lateral rectus muscles and
orbital width were measured to determine the horizontal
muscle index ([D+E/F]) (Figure 5). The greater of the two
ratios was considered the muscle index. The study showed
that a muscle index of 67% or greater indicated compressive
neuropathy with a diagnostic sensitivity of 67%, although
no patient with NOD had a muscle index of less than 50%.

Figure 6 - Coronal CT scan showing orbits with apical crowding
due to enlarged extraocular muscles. The right orbit shows
effacement of the perineural fat up to 25% of the circumference
(grade 1 in Nugent’s score); the left orbit shows no effacement of
the perineural fat (grade 0).

mechanism. The authors also observed reductions in the
optic nerve diameter in the absence of enlarged extraocular
muscles, suggesting that the compression results from
increases in the intraorbital pressure due to the increased
fat volume.
Although CT is excellent for outlining bone details, MRI
provides better soft tissue detail and is useful for evaluating
the extraocular muscles, optic nerve and fat. Therefore,
further studies are necessary to better investigate the ability
of MRI to detect DON, alone or in association with CT.
Ultrasonography and color Doppler imaging. In some
studies, US has been used to attempt to identify the
presence of DON. One study has suggested that US can
detect DON-related enlargement of the subarachnoid space
of the optic nerve, but this is apparently only rarely noted
(4). The evaluation of the venous flow can also be useful.
Monteiro et al. (66) reported that patients with congestive
orbitopathy and predominantly myogenic fibrotic GO
experience a significant reduction in the SOV flow, matching reports from other authors (81) and supporting the idea
that orbital congestion due to extraocular muscle enlargement can reduce orbital venous drainage. Because DON is
known to be related to orbital apex crowding, the existence
of severe venous stasis in the orbits possibly reflects a stage
in its development. Although both US and CDI can
produce findings suggestive of DON, systematic studies
differentiating congestive cases with DON from cases
without DON are needed to determine the usefulness of
these methods.
Although the diagnosis of GO and DON still relies heavily
on clinical data, recent decades have seen outstanding
developments in orbital imaging techniques. Important
imaging studies have led to the emergence of new perspectives in the diagnosis and treatment of GO, which constitute
the main topic of this review.
CT remains the main imaging modality in Gravesâ&#x20AC;&#x2122;
disease. CT can be used to establish the degree of extraocular muscle and orbital fat enlargement, clarify a confusing clinical picture and aid surgical planning. Furthermore, CT can be of great help in the detection of DON
using linear, area or volumetric indexes of orbital apex
crowding. However, CT provides little information on the
disease activity, except through the observation of changes
in sequential studies.
MRI can also be used to diagnose GO. However, the
ability of MRI to provide evidence for DON has still not
been explored, perhaps due to the difficulty of estimating
the orbital bone volume and thereby determining the
amount of orbital apex crowding. However, due to its
greater ability to differentiate soft tissues, MRI has become a
useful adjunct in imaging studies of GO patients when GOrelated muscle involvement must be differentiated from
other orbital conditions. Additionally, progressive technical
refinements will likely enhance its usefulness, especially in
the assessment of GO activity upon diagnosis and during
treatment.
US is a well-established, accessible and low-cost technique widely used to detect extraocular muscle enlargement.
However, the inability to perform an accurate evaluation of
the orbital apex compared with CT or MRI has reduced the
importance of US in the diagnosis and management of GO.
Although it is still in an early developmental stage, CDI may
become useful in the management of GO because CDI is the
technique that best evaluates orbital venous congestion, a

2, and 3 orbital crowding. More recently, the objective
quantification of apical orbital crowding based on square
area measurements was shown to be a more efficient
diagnostic tool than subjective quantification. In this study,
the best-performing index was highly efficient at detecting
DON, with a sensitivity of 91.7% and specificity of 90.0%
(75).
Chan et al. (73) highlighted the importance not only of
extraocular muscle enlargement but also the role of the bony
orbit and its usefulness as a predictor of DON. The bony orbit
capacity was quantified using standardized orbital angles on
axial scans. The study showed that these measurements were
independent predictors of DON and that narrower orbits
were more susceptible to develop DON. The authors also
calculated an index of orbital muscular crowding in
combination with lateral and medial orbital wall angles that
had a 73.3% sensitivity and 90% specificity.
Birchall et al. (74) found intracranial fat prolapse through
the superior orbital fissure to be a predictor of DON, but
later studies have not confirmed the usefulness of this
parameter (70,73). Other CT features have been tested as
indicators of DON, including lacrimal gland displacement,
exophthalmos, superior optic vein dilatation, and single
muscle measurements, yielding conflicting and mostly
discouraging results (9,17,32,52,74,76).
Although linear or square measurements have proved
helpful in diagnosing DON, the estimation of the orbital
apex crowding based on volumetric estimates of structures
could potentially improve the ability to detect DON.
Feldon et al. (14,77) have used volumetric estimation of
the orbital content to investigate the risk of developing
DON in GO. Previously, such estimates involved cumbersome measurements of the extraocular muscles, but recent
advances in MDCT have made volumetric estimates of
orbital structures readily available at a workstation (78). In
a recent study, orbital crowding indexes were for the first
time calculated based on the volumetric analysis of CT
images. The orbital fat and muscle volumes were estimated
based on their different attenuations in Hounsfield units
from measurements from the anterior orbital rim up to the
optic foramen. Based on these measurements, two volumetric indexes of orbital muscle crowding were calculated:
one based on axial scans of the entire orbit and another
based on coronal scans of the orbital apex. Both indexes
were efficient in predicting DON, especially the index
limited to the orbital apex (with 92% sensitivity and 86%
specificity) (79).
Magnetic Resonance Imaging (MRI). Very few MRI
studies have directly assessed DON. Dodds et al. (80)
assessed DON in a high-resolution MRI study in which
they compared the diameter of the optic nerve at seven
different positions from the eyeball to the pre-chiasmal
region in three groups (control, GO with DON, and GO
without DON). The optic nerve diameter was significantly
smaller in the group with DON. However, the overall
reduction was not substantial, and there was considerable
overlap between the control subjects and patients with DON.
The authors admitted that despite the observed reduction in
the optic nerve size in the DON group, neural compression
could not be shown to be the pathogenic mechanism
underlying the nerve dysfunction, but they found
mechanical compression of the optic nerve to be the most
plausible explanation. Alternatively, DON might be caused
by vascular compression or some other unidentified

1332

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Imaging studies in Graves’ orbitopathy
Gonc¸alves ACP et al.

possible contributing factor during the active congestive
stage of GO.

27.

AUTHOR CONTRIBUTIONS

28.

Gonc¸alves AC contributed to the review of the literature and writing of the
manuscript. Gebrim EM revised the manuscript. Monteiro ML wrote and
revised the manuscript.

Airway diseases are highly prevalent worldwide; however, the prevalence of these diseases is underestimated.
Although these diseases present several common characteristics, they have different clinical outcomes. The
differentiation between asthma, chronic obstructive pulmonary disease and bronchiectasis in the early stage of
disease is extremely important for the adoption of appropriate therapeutic measures. However, because of the high
prevalence of these diseases and the common pathophysiological pathways, some patients with different diseases
may present with similar symptoms. The objective of this review is to highlight the similarities and differences
between these diseases in terms of the risk factors, pathophysiology, symptoms, diagnosis and treatment.
KEYWORDS: Asthma; Chronic Obstructive Pulmonary Disease; Bronchiectasis.
Athanazio R. Airway disease: similarities and differences between asthma, COPD and bronchiectasis. Clinics. 2012;67(11):1335-1343.
Received for publication on September 27, 2012; Accepted for publication on September 27, 2012
E-mail: rathanazio@yahoo.com.br
Tel.: 55 11 2661-5695

in smokers, 7% in former smokers and 3% in individuals
who never smoked (3). Surveys on the prevalence of asthma
suggest a prevalence of approximately 9% in the British
population. In Brazil, an epidemiological study of the
population in Sa˜o Paulo revealed a COPD prevalence of
15.8% (4), whereas the prevalence of asthma was estimated
to be approximately 10% of the general population (1).
Several studies have suggested a similar prevalence of
asthma among children and adult populations; however,
extensive variability has been found depending on multiple
factors that include geographic differences and socioeconomic status (5).
Because of a lack of well-conducted epidemiological
studies, an accurate prevalence of bronchiectasis is more
difficult to estimate than that of asthma and COPD. There
has been a decrease in the incidence of this disease, which
has been attributed to the increased use of antibiotics for
infection control and immunization strategies in children.
Tsang and Tipo (6) reported a hospital admission rate of
16.4 per 100,000 people and a mortality rate of 1 out of
100,000 people in Hong Kong.
The overlap in the terminology that is used to define
asthma, chronic bronchitis, emphysema, COPD and bronchiectasis is the greatest cause of confusion in distinguishing
these diseases and in accurately determining the prevalence
of airway diseases. The prevalence of obstructive diseases in
adults can vary by more than 200% in the general
population and depends on the definition that is used (a
self-reported diagnosis versus a diagnosis based on spirometry findings) (7). Furthermore, the actual prevalence of
obstructive diseases is underestimated. When a spirometric
evaluation was performed in the general population,
approximately 58% of the patients with an obstructive
disorder did not report a prior lung disease diagnosis (8).
Individual patients may have different combinations of
airway diseases. Studies in an American population

INTRODUCTION
The prevalence of airway diseases has increased in recent
decades despite therapeutic advances. Furthermore, the
prevalence of these diseases is underestimated according to
epidemiological surveys, which further increases the complexity of managing these diseases. In Brazil, acute asthma
exacerbations and chronic obstructive pulmonary disease
(COPD) are major causes of hospitalization (1,2). Despite the
presentation of similar symptoms, such as dyspnea, coughing, wheezing and expectoration, airway diseases have
different underlying pathophysiological processes and must
be distinguished to enable the administration of appropriate
treatment. With an appropriate clinical history and objective
diagnostic testing, the distinction between these diseases
can be performed efficiently in most cases. However, many
patients who are evaluated for respiratory symptoms are
misdiagnosed due to an atypical case presentation, an
insufficient etiological investigation or an overlapping of the
diseases.
This review aims to present the similarities and differences between airway diseases and suggest a practical
approach for the differentiation of the most common
respiratory illnesses, i.e., asthma, COPD and bronchiectasis.

Epidemiology
Because of the variability in the definition of COPD in
epidemiological studies, an accurate prevalence of this disease
is difficult to determine. The prevalence is approximately 14%

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
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No potential conflict of interest was reported.

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several risk factors have been found, including smoking,
occupational exposure, advanced age, an unfavorable socioeconomic condition and housing in an urban center (18).
Asthma can be induced by either animal or plant proteins
and by organic and inorganic chemical agents (19,20). In
addition, the development of asthma is correlated with the
Western lifestyle, which is characterized by a high hygiene
rate. Increased hygiene reduces exposure to allergens and
decreases natural desensitization (21). The exposure to
external agents can result in the development of COPD,
which is mainly associated with occupational activities,
such as coal and gold mining (22), cadmium mining (23)
and exposure to smoke from burning wood (24,25).
Regarding bronchiectasis, exposure to fungi is a cause of
exacerbated allergic responses, such as in allergic bronchopulmonary aspergillosis (15).
Smoking is the main etiological factor for the development of COPD. While it is known that quitting smoking is
the only factor that can slow the progression of this disease,
there is no evidence that there exists a reversion for
the pulmonary impairment that has already began.
Approximately 90% of COPD cases are related to smoking,
whereas other less common risk factors include occupational exposure and biomass burning (26,27,28). The
historical finding of a lower prevalence of COPD in the
female population is associated with a lower proportion of
smokers in this group. However, an increase in the number
of women who smoke in recent decades has increased the
prevalence of COPD in the female population and the
mortality that is associated with this pathology (29-31).
In Brazil, data indicate a decline in the smoking
prevalence (32); however, several factors are extremely
relevant. There are an insufficient number of effective public
policies that discourage smoking among young people who
are usually influenced by alcohol consumption, media
advertising and paternal smoking (33). In addition, a large
portion of pneumologists in Brazil need to be trained
because these physicians cannot effectively treat smoking
(34). These steps are crucial in reducing the prevalence of
COPD.
Regarding genetic factors, several conditions are classically associated with the development of COPD. A
deficiency of alpha-1 antitrypsin decreases the defense of
the lungs against inhaled noxious agents, thus increasing
the development of emphysema. Patients with this deficiency account for approximately 1-2% of COPD cases

demonstrated that more than 15% of the patients with an
obstructive disease received more than one diagnosis, and
this rate reached 50% in a population older than 50 years of
age (8). In Australia (9), this proportion was approximately
25% in individuals who were between 45 and 69 years of
age. In Italy (10), approximately 20% of the asthmatic
population had symptoms that included a productive
cough, which is compatible with a diagnosis of chronic
bronchitis. Patients who exhibit the coexistence of two or
more obstructive diseases tend to be older and have
spirometric data that indicate lower values of forced
expiratory volume in the first second (FEV1) (8).
Furthermore, the coexistence of asthma and COPD was
associated with a higher mortality rate (11).
The high prevalence and morbidity of these diseases
translates into a substantial cost to the healthcare system.
Drug costs are the main expenses that are associated with
the treatment of asthma, whereas COPD and bronchiectasis
have a greater economic impact due to high hospitalization
rates (12). The main findings from these epidemiological
studies are as follows: (1) the prevalence of chronic
obstructive pulmonary disease, which is closely correlated
with the definition that is used, is an important social and
economic problem; (2) the overlap between asthma, COPD
and bronchiectasis is associated with an increase in clinical
severity and mortality; and (3) approximately half of
patients with obstructive findings on spirometry are not
properly diagnosed; therefore, screening programs that
include a pulmonary function evaluation (with an assessment of both spirometry and peak flow) should be adopted
to decrease the proportion of patients without adequate
monitoring (13,14).

Risk factors
Patients with asthma and COPD can usually be distinguished according to the classic risk factors that are
associated with each disease. However, certain risk factors
may be common in both diseases. Regarding the illnesses
that are associated with bronchiectasis, the identification of
the risk factors is crucial and is complex because of the wide
variety of conditions that predispose a patient to a
permanent dilation of the airways (15) (Table 1).
In atopic individuals, the main risk factor for developing
asthma is exposure to allergens. Consequently, many
patients with asthma have high serum levels of IgE and
eosinophils (16,17). For individuals with non-atopic asthma,
Table 1 - Risk factors for asthma, COPD and bronchiectasis.

membrane thickening. These alterations are positively
correlated with the frequency of asthma attacks and
bronchial hyperresponsiveness (55,56).
COPD patients exhibit a reduced airway caliber, which is
associated with cell damage that is induced by external toxic
agents, especially cigarette smoke, via reactive oxygen
species (57,58). The presence of goblet cells (mucous
metaplasia) in the small airways and mucous hypersecretions results from the process of airway narrowing (59,60).
Despite the prevalence of inflammation in COPD, which
occurs due to the presence of neutrophils and macrophages,
several studies have demonstrated the presence of eosinophilic inflammation both in stable patients and in patients
with acute exacerbations of the disease (61). This finding
confirms the potential anti-inflammatory effect of inhaled
corticosteroids in the treatment of COPD. Another interesting finding for this disease is the correlation between
inflammation intensity and COPD severity. In the final
stages of the disease, an intense inflammatory process
occurs, which suggests that, even in these scenarios,
treatment with anti-inflammatory drugs, such as inhaled
corticosteroids, may be effective (62).
Bronchiectasis develops with recurrent damage to the
airways, which generally occurs in individuals with
mucociliary clearance that is altered by genetic susceptibility, thus leading to inflammation and destruction of the
muscular and elastic components of the bronchial walls (63).
Respiratory infections are the leading causes of bronchiectasis; however, other pro-inflammatory attacks can trigger
or accelerate the process, such as a toxin inhalation,
environmental exposure, smoking, aspiration of gastric
contents or changes in immune responses (6).
Abnormally dilated airways are susceptible to bacterial
colonization, which leads to a constant presence of
inflammation that is mainly mediated by neutrophils (64).
There is progressive impairment of ciliary function with a
worsening of airway mucociliary clearance, which further
facilitates the presence of bacterial colonization and the
accumulation of thick mucus (65,66). In addition, the
perpetuation of inflammation leads to further damage to
the mucosal integrity of the airways, thus promoting
continuous bacterial invasion and permanence in the
mucosa. This infectious and inflammatory cyclic process
causes progressive damage to the bronchial wall with
associated clinical deterioration. Increased arterial bronchial
proliferation and arteriovenous malformations can occur as
a result of the inflammatory process and bronchial wall
alterations. This vicious cycle can produce significant
bacterial proliferation and inflammation with increased
suppuration and clinical worsening.
However, understanding the inflammatory patterns of
each disease is important for distinguishing between airway
diseases. In a study of 27 COPD patients and 19 asthma
patients with similar degrees of pulmonary obstruction,
several parameters were evaluated. Several functional
(residual volume and diffusion capacity) and tomographical
(emphysema score) parameters could differentiate between
the individuals with COPD and those with asthma.
However, the inflammatory and pathological features of
basal membrane thickening, eosinophilia and the CD4/CD8
relationship in the bronchoalveolar lavages were the best
predictors of a history of asthma (67).
Immunological differentiation may have an important prognostic role in patients. Sputum eosinophilia is

(35,36). Other genetic influences on the development of
COPD include a polymorphism in the promoter region of
inflammatory mediators, such as tumor necrosis factoralpha (TNF-alpha) (37), and polymorphic variants in the
hydrolase-encoding genes (38).
Several genetic diseases are associated with the development of bronchiectasis. Cystic fibrosis is characterized by a
mutation in the cystic fibrosis transmembrane conductance
regulator (CFTR) gene and is the leading cause of genetic
disease-related death among Caucasians, which is typically
due to respiratory failure. In addition, immotile cilia
syndrome (primary ciliary dyskinesia) and genetic anomalies that are associated with a humoral or cellular
immunodeficiency are common causes of bronchiectasis
(39).
Several studies have analyzed the development of asthma
and specific genetic alterations but have not found an
association. Because of the multifactorial presentation of this
disease, the existence of a single genetic site associated with
the development of this disease is questionable. However,
evidence suggests that chromosomal regions may modulate
the degree of disease severity, such as the relationship
between chromosome 2q and the levels of IgE and bronchial
hyperresponsiveness (40).
The influence of gender on the development of asthma
varies with age. Childhood asthma is more common among
boys, whereas women are more commonly diagnosed with
asthma in adulthood (41,42). COPD is more common among
men than women, which is related to the gender difference
in smoking intensities (43). However, women develop more
severe airflow obstruction than men after an adjustment for
the tobacco intake intensity (44,45).
Additionally, other factors influence obstructive respiratory diseases. The presence of gastroesophageal reflux is
correlated with increased inflammation in the airways of
patients with bronchiectasis and is associated with
increased asthma severity. However, because of a lack of
well-conducted longitudinal studies, the relationship
between gastroesophageal reflux disease and bronchial
hyperresponsiveness may not be causal and may not be
associated with severity (46). In addition, a low birth weight
may predispose individuals to the development of asthma
(47) or COPD (48). The proposed mechanism for this
association is based on the normal respiratory functional
decline with age that occurs from a lower peak in these
individuals. Additionally, a history of viral or bacterial
infections in childhood is correlated with the development
of asthma, and these infections are well-established causes
of bronchiectasis in adulthood (49,50). Passive smoking and
a deficiency of certain dietary elements, such as polyunsaturated fatty acids, are associated with the development of
chronic airway inflammation in adulthood (51).

Physiopathology
Asthma, COPD and bronchiectasis are diseases that cause
chronic inflammation of the airways but have distinct
characteristics. In asthma, eosinophils, mast cells and CD4
T lymphocytes represent the predominant cell types in the
inflammatory process. In contrast, COPD and bronchiectasis
demonstrate a greater number of neutrophils, macrophages
and CD8 T lymphocytes (52-54).
Asthmatic patients have airway obstructions that are
predominantly characterized by bronchoconstriction
through the activation of the smooth muscle and basal

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CLINICS 2012;67(11):1335-1343

change in dyspnea, cough and/or basal expectoration of the
patient who goes beyond the normal daily variation, and
that can cause a change in regular medication of the
patient’’ (78).

associated with difficult-to-control asthma (68), and the
normalization of this condition after treatment was correlated with a decrease in the number of exacerbations and
hospitalizations. Neutrophilic inflammation in asthma is
less sensitive to corticosteroid treatment and is associated
with rapid functional loss (69).
Despite the distinct pathophysiological mechanisms of
airway diseases, these diseases share several common
features. Specific therapies, especially anti-inflammatory
medications and bronchodilators, may be the most beneficial treatments for these diseases because these drugs
control symptoms and improve the quality of life of the
patients.

Lung function
In asthma, the typical finding of airflow obstruction that
is characterized by a decrease of VEF1/FVC (forced vital
capacity) with a disorder reversal after the administration of
a bronchodilator is the mainstay in the diagnostic confirmation of the disease (79). However, many patients may
present with a reversal after the use of a bronchodilator
without exhibiting normalization in pulmonary function
tests, thus indicating signs of bronchial remodeling. In
addition, clinically stable patients may present with normal
spirometry but a positive bronchial provocation test (1).
Other functional variables can be used for the diagnosis of
obstructive lung disease, such as the forced expiratory flow
at 50% of the FVC FEF50%/0.5FVC (80) ratio. However, the
use of the FEV1/FVC ratio remains the most widely
accepted parameter for diagnostic confirmation.
In COPD, a post-bronchodilator FEV1,80% that is
associated with a FEV1/FVC ratio ,70% confirms the
obstructive disorder, which is characterized by a lack of
complete reversibility of airflow. However, a significant
response to the bronchodilator does not exclude a COPD
diagnosis (78).
More accurate functional tests can differentiate between
these two diseases through measures that assess lung
hyperinflation and the diffusion capacity of the lungs for
carbon monoxide (DLCO). Hyperinflation, which is confirmed by plethysmography, is more commonly found in
COPD that is characterized by a greater residual volume
than is found in asthma. Another characteristic of COPD is a
decrease in the DLCO (70).
Early in the course of the disease, patients with
bronchiectasis usually present with lung function tests that
are characteristic of an obstructive disorder, thus confirming
the inflammatory nature of this disease and the initial
involvement of the small airways. However, with the
progression of the disease, a mixed functional disorder or
a restrictive disorder can be found due to the progressive
destruction of the pulmonary parenchyma, which is
characterized by recurrent infectious exacerbations and
massive destruction of the small airways (81).

Diagnosis
Characterizing the physiological and phenotypic differences between patients with obstructive diseases is important for obtaining a greater understanding of the evolution
of these diseases and the therapeutic implications. Most
patients can be distinguished by a detailed clinical history
and simple functional and imaging tests; however, many
patients have atypical clinical profiles due to the heterogeneity of the airway diseases, which can lead to a
misdiagnosis. This factor is particularly important for the
elderly population in which the coexistence of asthma,
COPD and bronchiectasis is common.

Symptoms
Asthmatic patients who exhibit bronchoconstriction are
characterized by wheezing, breathlessness, chest tightness
and coughing. The characteristic of the reversible inflammatory process in asthma and a good response to
therapeutic measures characterize the evolution of the
disease, which is marked by intermittent exacerbations
(70-72). However, the lack of these symptoms can define a
subgroup of patients who are considered hypoperceivers
and who evolve with a worse prognosis for disease control
(73).
The main clinical manifestation of patients with bronchiectasis is a chronic cough with sputum production,
although patients may only have a dry cough. Patients
often have dyspnea on exertion (75%), wheezing (75%) and
pleuritic chest pain (50%). Additionally, patients may have
systemic symptoms, such as fatigue or weight loss, and
rhinosinusitis. A physical examination may identify crackles
(70%), snoring (44%) and wheezing (34%). Clubbing
appears in approximately 3% of the cases. The clinical
evolution consists of a progressive functional loss and is
marked by recurrent infectious exacerbations with the need
for frequent antibiotics (15).
Patients with COPD have a cough that is frequently
associated with chronic sputum production (74). Characteristically, these individuals present with dyspnea and
effort limitations due to the fixed airflow obstruction (7577). More advanced stages of the disease result in a worse
quality of life for these patients. Respiratory exacerbations
are associated with an increased underlying inflammatory
process that requires appropriate therapy with antibiotics
and systemic corticosteroids. These exacerbations are
characterized by a worsening of the basal symptoms of
the patient, such as cough, expectoration and dyspnea.
According to the Global Initiative for Chronic Obstructive
Lung Disease (GOLD), an exacerbation is defined as ‘‘an
event in the natural course of the disease characterized by a

Imaging tests
Chest radiography is not sufficiently sensitive for the
diagnosis of airway diseases, and this test is recommended
for the differential diagnosis of a patient with respiratory
symptoms. In more severe cases of patients with bronchiectasis, the dilation of large airways can be visualized as a
thickening of the peribronchovascular interstitium using
this method. A universal radiographic finding in obstructive
diseases is lung hyperinflation, which is characterized as
follows: (1) an increase in the lung volume, (2) an increase in
the intercostal spaces, (3) a rectification of the diaphragmatic
domes, (4) an accentuation of the retrosternal space and (5)
the presence of air below the inferior border of the heart
(82).
A thoracic CT scan is a more sensitive test than chest
radiography and is useful in the management of airway
diseases. This test is considered the gold standard in the
diagnosis of bronchiectasis. Common findings in obstructive

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Differentiating airway diseases
Athanazio R

diseases include bronchial wall thickening, centrilobular
nodules and mosaic attenuation by air trapping (82).
The following tomographic criteria are used for the
diagnosis of CT bronchiectasis: (1) a bronchial internal
diameter that is larger than 1.5 times the diameter of the
adjacent pulmonary artery (signet ring sign), (2) an absence
of a gradual decrease in the bronchial diameter from the
central regions to the periphery and (3) bronchial visualization in the periphery 1-2 cm from the parietal pleura (6).
According to CT scan findings, we can classify bronchiectasis as cylindrical (bronchial dilation), varicose (with focal
constrictions along the airways) or cystic (saccular dilations
at the end of a bronchus). In addition, we can classify the
bronchiectasis as localized (i.e., confined to one lobe) or
generalized (83).
In asthma patients, bronchial wall thickening and air
trapping are commonly found; however, patients with mild
asthma may have completely normal thoracic tomography
findings. However, current analyses with quantitative
techniques and high-resolution scans can distinguish
between normal and mildly controlled asthmatic individuals
based on the degree of air trapping and bronchial thickening.
Moreover, severe asthma is associated with a greater degree
of bronchial thickening in these individuals. Another interesting finding of these studies is that a greater degree of air
trapping was associated with an increased risk of exacerbation. Because CT scan is noninvasive, this technique may be
used to monitor patients with severe asthma, especially for
the evaluation of bronchial remodeling (84,85).
In patients with COPD, the most important tomographic
finding is the presence of centrilobular emphysema in the
superior lung fields (smoking-related) or diffuse panlobular
emphysema (alpha-1 antitrypsin deficiency). The intensity
of emphysema, which is characterized by the amount of
area with low attenuation, and bronchial thickening
correlate with the degree of airflow obstruction that is
measured in functional tests (86-88).
Therefore, thoracic tomography is an important tool that
can be used in the differential diagnosis of airway diseases
(Figure 1). However, many of these CT findings are
common among several obstructive diseases, and more
advanced stages of asthma and COPD may lead to the
development of bronchiectasis.
The diagnosis of an airway disease in patients should
begin with a detailed anamnesis, and complementary exams
should be performed as appropriate. No single test can
completely differentiate between these diseases. The coexistence of more than one disease in the same patient should
always be considered when a patient presents with an
unfavorable clinical evolution or when their laboratory tests

are not consistent with the initial clinical hypothesis
(Table 2).

Treatment
Asthma treatment guidelines aim for the appropriate
control of symptoms through a strategy of phased measures
that focus on the severity of the disease and the daily
complaints of the patient. Once this goal has been achieved,
the treatment should be maintained at the lowest possible
dosage to reduce side effects and the associated costs (1,5).
In addition, the guidelines for the treatment of COPD use a
phased strategy according to the severity of the disease.
However, the guidelines emphasize the prevention of
disease progression. Moreover, once the treatment goal
has been achieved, a reduction in the medication dosage is
uncommon (2,78,89). Regarding bronchiectasis, there is a
lack of literature on the guidelines for the treatment of this
pathology. British guidelines were recently published (90);
however, there are few data on the therapeutic recommendations for bronchiectasis due to a lack of well-conducted
randomized clinical trials. Many measures are still being
extrapolated from studies of cystic fibrosis, but physicians
must be aware of the differences between the various
diseases that may be similar to bronchiectasis (91).
Much of the therapeutic arsenal is common between
obstructive diseases, especially bronchodilators (beta2-agonists and anticholinergics) and inhaled corticosteroids.
However, the treatment goal for each disease may vary.
COPD therapy is directed primarily to the relief of
symptoms and the prevention of disease progression. In
bronchiectasis, the primary goal of treatment is to prevent
disease progression and improve the quality of life and
symptoms. In asthma, the primary goal of treatment is to
control the underlying inflammatory process with the
consequent control of symptoms.

Beta2-agonists
Bronchodilators with a direct action on beta-adrenergic
receptors can be classified as short- or long-term depending
on the half-life.
Short-acting beta2-agonists (SABAs) are the first-line
treatment for COPD; however, the use of SABAs as a rescue
medication in asthma is appropriate. In patients with
bronchiectasis, SABAs are generally used for symptom
relief despite the lack of evidence for the use of these drugs.
In contrast, long-acting beta2-agonists (LABAs) are used
in combination with anti-inflammatory medications in
asthma. LABAs may be used alone in patients with
COPD. The isolated use of a beta2-agonist in asthma is
contraindicated and is associated with a poor prognosis (92).

Asthmatics Smokers have an
increased risk of developing COPD
Asthma and bronchiectasis are
misdiagnosed in the elderly and
are commonly mistaken for COPD
Patients with bronchiectasis are
diagnosed late because they are
first treated for COPD due to
productive cough symptoms
Asthmatic patients may lose
reversibility over time
Bronchial thickening can occur in
patients with COPD and
bronchiectasis, and bronchiectasis
may appear in asthmatics and
individuals with COPD

Risk factors
Age

Computerized tomography

COPD: Chronic obstructive pulmonary disease.

These drugs act by relaxing the bronchial smooth muscle,
increasing the mucociliary clearance, decreasing the vascular permeability and possibly reducing inflammatory
mediators (5).
In COPD, the use of LABAs is associated with functional
improvement (increased FEV1), symptom control and
improved quality of life (93,94). In asthmatic patients, the
combination therapy of a LABA with inhaled corticosteroids
(ICs) is more efficient in controlling symptoms than an isolated
increase in the IC dosage (95). For patients with bronchiectasis,
the combination of a LABA with a conventional IC improved
the symptom scores and quality of life (96).

asthma, inhaled corticosteroids are the mainstay of disease
treatment and are considered the first-line treatment in
patients with persistent asthma. ICs can reduce the number
of exacerbations, improve lung function and quality of life,
control respiratory symptoms and decrease the bronchial
hyperresponsiveness (102-104). Current guidelines emphasize the use of an IC in the initial therapy of patients with
asthma symptoms. The combination of an IC with a LABA
is recommended as the next step in therapy despite the
potential need to increase the IC dosage (1,5). However, the
use of measures that increase patient adherence to treatment
and the adequate use of inhalers are fundamental to
achieving good clinical control of symptoms (105-108).
The use of an IC to treat COPD was effective in reducing
airway inflammation, although the IC did not affect the rate
of functional decline. There are conflicting data on the
ability of an IC to improve lung function, whereas there are
concrete data that associate the use of an IC with a
significant reduction in the exacerbation rate of COPD and
improvements in the quality of life of COPD patients (109).
The discontinuation of treatment results in a poor quality of
life and a faster exacerbation recurrence (110). Current
guidelines recommend the use of an IC in patients with
advanced pulmonary disease that is characterized by a
severe obstructive disorder or a large number of exacerbations (78).
In patients with bronchiectasis, the use of an IC was
effective in reducing the inflammatory markers and sputum
volume. The amount of daily sputum in patients with
bronchiectasis is an important severity marker that correlates with the number of lung exacerbations. However,
studies have not demonstrated a significant impact of ICs on
the lung function and exacerbation rate in individuals with
bronchiectasis (111).

Anticholinergics
The release of acetylcholine by vagal stimulation triggers
a bronchoconstrictor response and an increased production
of pulmonary secretions. The use of short-acting anticholinergic drugs (ipratropium) in patients with COPD
yielded a dose-dependent functional improvement with
better responses than those that were achieved using a
SABA. For asthma, ipratropium is usually only used for the
management of severe asthma attacks, and there is no
evidence of benefits in patients with bronchiectasis (97).
Long-acting anticholinergics (LAMAs) have a prolonged
half-life (36 hours) with an action peak of approximately 1-2
hours (98). Tiotropium is the most studied pharmacological
agent, and COPD studies have indicated satisfactory results,
including a reduction in the number of exacerbations, an
improvement in quality of life and an increase in the FEV1
(99). There is no evidence that supports the superiority of
LAMAs over LABAs; however, the combination of these
drugs may be used with an additive effect in patients with
accentuated lung function or significant functional limitations (100). Recently, the use of tiotropium in asthma has
been studied as an alternative in patients with contraindications to the use or in combination with LABAs (101).
Similar to ipratropium, the use of tiotropium for the
treatment of bronchiectasis has not been adequately studied.

Other pharmacological agents
Xanthines, such as theophylline, have moderate bronchodilator effects, immunomodulatory properties and antiinflammatory effects, which increase sensitivity to corticoids
in the nucleus of inflammatory cells through the histone
deacetylase pathway. However, the clinical effects of
xanthines, which are associated with a reduced therapeutic
range and an increased risk of severe side effects, have
reduced the clinical applicability of these drugs. The use of

Corticosteroids
The underlying inflammatory process in obstructive
diseases has always been the focus of therapeutic interventions that aim to reduce disease progression, improve lung
function and reduce symptoms and exacerbations. In

xanthines is increasingly reserved for severe cases, especially in patients with COPD (78).
Anti-leukotrienes, such as montelukasts, can reduce
eosinophilic inflammation through inhibition of the lipoxygenase pathway. The bronchodilator effect of anti-leukotrienes is discrete, and the use of these drugs is reserved for
asthmatic patients; however, there is no concrete clinical
applicability of these drugs in COPD (5).

Other nonpharmacological therapies
Nonpharmacological measures are important in the
management of patients with an obstructive disease.
Smoking cessation in patients with COPD or who are at
risk for the development of COPD (such as individuals with
asthma) must be encouraged. Although lung damage can
continue to progress, smoking cessation was effective in
reducing the rate of lung function decline (112).
Viral infections are a major cause of exacerbations in
patients with asthma, COPD and bronchiectasis and are
associated with a higher mortality rate. Vaccination against
influenza is demonstrably linked to a reduction in severe
exacerbations and mortality. Therefore, all patients with an
obstructive disease should be directed to receive an annual
flu shot. Conversely, the efficacy of the pneumococcal
vaccine is not yet fully understood. Patients with a
respiratory disease are at an increased risk of hospitalization
for pneumonia, especially the elderly; therefore, the use of
this vaccine should be considered (5,78).
The use of bronchial thermoplasty has been considered an
option in patients with severe asthma. Studies in individuals with severe persistent asthma demonstrated a reduction in the exacerbation rate and an improvement in
symptom control. The finding of hypertrophied smooth
bronchial muscles in asthma patients justifies the use of this
approach. The use of a probe by bronchoscopy that releases
heat into the airway and leads to the destruction and/or
atrophy of bronchial smooth muscle supports the biological
plausibility for the use of this technique (113,114).
Pulmonary rehabilitation and an increase in physical
activity interventions are useful for the improvement of
respiratory symptoms and fitness. These measures are
effective in the treatment of COPD patients (115). In addition,
data in the literature suggest that asthma and bronchiectasis
patients may benefit from these measures (116).
The prevalence of obstructive diseases continues to
increase worldwide, with a considerable social and economic impact. Understanding the pathophysiological processes that underlie the various diseases that cause airflow
limitations is essential for differentiating between these
diseases. Therefore, specific diagnostic methods can be
used, and adequate therapeutic interventions can be
applied. However, the possibility that more than one
condition coexists in the same patient should not be
underestimated, especially due to the high prevalence of
these diseases in the elderly population.

ACNOWLEDGMENTS
I would like to thank my colleagues Maria Cecı´lia Nieves Teixeira Maiorano
and Daniel Antunes Pereira for their contribution in reviewing this work.

(9). Tualang honey also exerts anti-oxidant activities against
pancreatic cells, thus reducing hyperglycemia in diabetic
models (10).
The above examples clearly illustrate the various potential
uses of tualang honey and the need for further studies to
fully elaborate the extent of its properties.

I read the recent article by Zaid et al. (1) with great interest.
Recent research has shown that tualang honey may have a
number of systemic benefits in addition to its protective effect
on bone structure in post-menopausal animal models.
Tualang honey has considerable potential as an anti-cancer
agent. For example, it exerts anti-proliferative activities against
breast cancer tissue, attenuating tumor growth in MDA-MB231 and MCF-7 cell lines (2). These anti-neoplastic effects are
mediated by caspase 2 and caspase 9 activation and a
reduction of the mitochondrial membrane potential in cancer
cells, reflecting an increase in apoptosis. Tualang honey
administration also produces early apoptosis in osteosarcomas
in a dose-dependent manner (3) and attenuates proliferation in
HeLa cell lines (2). Apoptosis is also enhanced in oral
squamous cell carcinomas following exposure to tualang
honey (3).
Furthermore, tualang honey reduces photo-carcinogenesis secondary to ultraviolet B radiation exposure (4). These
anti-carcinogenic effects are mediated by an attenuation of
PGE-2 synthesis and inhibition of the nuclear translocation
of NF-kB in keratinocytes. Methanol extracts of tualang
honey also decrease proliferation in keloid fibroblasts and
may thus be of clinical use in the dermatological treatment
of keloids (5). Interestingly, gamma radiation enhances the
anti-oxidant potential of tualang honey (6).
Tualang honey is considered by some to be the natural
equivalent of ‘‘hormone replacement therapy’’. For example, short-term memory is improved in post-menopausal
women following the administration of tualang honey (7),
which is comparable to the increase in short-term memory
observed after the administration of estrogen/progesterone
combination therapy. The administration of tualang honey
also attenuates atrophy in uterine tissue and increases
vaginal epithelium thickness (8). It is also associated with a
lower post-menopausal increase in body weight.
Tualang honey also decreases the wound size of burns and
provides enhanced control and containment of burn infections, especially by bacteria such as Pseudomonas aeruginosa

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

The recently published article on cerebral sparganosis
was very interesting (1). Indeed, Wang et al. described a
relevant case study of sparganosis, which is an uncommon
tapeworm infection that is sporadically reported and can be
found in many tropical countries. Cerebral sparganosis has
been documented in Thailand (2), and it is typically initially
identified by brain imaging. One important concern
associated with this condition is the migratory path of the
parasite. Indeed, migration is not part of the common course
of infection with this parasite, and migration is more
commonly observed in other parasitic infestations, such as

gnathostomiasis. Of interest, sparganosis can become
manifest in the brain, and concurrent infection may also
be possible.

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.

the EGFR mutation frequencies. That study has a patient
selection bias. Despite the authors’ assertion that the study
sample represents all five Brazilian geographic regions, not all
of the NSCLC patients had the opportunity for EGFR mutation
assessment in their origin centers. The data were obtained from
the laboratory files, and this may be a source of bias. Among
the 63 patients with an EGFR mutation, 57 (90.04%) patients
presented with an adenocarcinoma histological type. In
previous studies, the rate of EGFR mutation in the adenocarcinoma histological type was 49% (5). The gender distribution in
this study is another point of controversy. In the Brazilian
study, the proportion of male patients was 57.97% (120/207),
which was less than 75%, the previously published proportion
of male patients (2-4). Differences in histology and gender
distribution may lead to an unexpectedly higher frequency of
EGFR mutation in the Brazilian population (30.4%) than has
been reported in other publications (16.6–16.9%) (3,6) because
the adenocarcinoma histological type predominates in this
patient selection (1-3). As in any retrospective study, several
potential sources of bias cannot be ruled out, and the reader
should take this into consideration when reading the manuscript. Despite these limitations, this study is important to
characterize the frequencies of EGFR mutations among
different populations and improve the systemic treatment
selection for patients with advanced NSCLC.

Dear Editor,
I read the recent article by Bacchi et al. in a recent issue of
your esteemed journal with great interest (1). The article is
highly thought provoking. Over the past few years, new data
have emerged that reveal the emerging role of epidermal
growth factor (EGF) and its receptor (EGFR) in the personalization of non-small cell lung cancer (NSCLC) treatment.
Currently, EGFR tyrosine kinase inhibitors (TKIs), such as
erlotinib and gefitinib, have been demonstrated to be
effective in prolonging progression-free survival (PFS) and
overall survival (OS) in patients with advanced NSCLC and
EGFR mutations in exon 19 and 21 (2). In 2009, Rosell et al.
reported EGFR mutations in 16.6% of lung cancer patients in
a Spanish study (3). In that study, the median PFS and
median OS were 14 months and 27 months, respectively (3).
A Japanese study conducted by Tanaka et al. confirmed that
EGFR mutations are more frequent in the adenocarcinoma
histological type and in light smokers (4). In Tanaka’s study,
the overall frequency of EGFR mutations was 31% (4).
Kosaka et al. conducted a study in Japan in 397 patients with
lung adenocarcinoma who underwent potentially curative
pulmonary resection (5). They found that 196 patients (49%)
had EGFR mutations. Of these, 83 mutations were exon 19
deletions (42%), and 92 were L858R (47%). The study (5)
showed that patients with EGFR mutations survived longer
than those without mutations (p = 0.0046). There was no
difference in the overall survival between patients with an
exon 19 deletion and those with L858R (p = 0.41) (5).
In the Brazilian study conducted by Bacchi et al., the EGFR
mutation frequency was 30.4% (1). In that study, 169 of 207
patients (81%) had adenocarcinoma, and 38 of 207 (18.35%)
patients had a non-adenocarcinoma histology. Of the 207
patients, 120 (57.97%) were male. EGFR mutations were more
prevalent in the adenocarcinoma histological type and in nonsmokers than in non-adenocarcinoma histological types and
smokers, respectively. The Brazilian study did not show
differences between Asian and non-Asian patients regarding

Copyright ß 2012 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.